WO2003034980A2 - A novel pharmaceutical compound containing abacavir sulfate and methods of making and using same - Google Patents

Abstract

A composition comprising a polypeptide and benzatropine mesylate covalently attached to the polypeptide. Also provided is a method for delivery of benzatropine mesylate to a patient comprising administering to the patient a composition comprising a polypeptide and benzatropine mesylate covalently attached to the polypeptide. Also provided is a method for protecting benzatropine mesylate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of benzatropine mesylate from a composition comprising covalently attaching it to the polypeptide.

Description

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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ABACAVIR SULFATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to abacavir sulfate, as well as methods for protecting and administering abacavir sulfate. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Abacavir sulfate is a known pharmaceutical agent - a carbocyclic 2'- deoxyguanosine nucleoside analogue that is a reverse transcriptase inhibitor used in the treatment of HIV. Its chemical name is (lS,4R)-4-[2-ammo-6-(cyclopropylamino)-9H- purin-9-yl]-2-cyclopentene-l -methanol. Its structure is as follows:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absorbtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW001P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absorption of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW001P

shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drug conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW001P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absorption through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (abacavir sulfate) to a polymer of peptides or amino acids. The invention is distinguished from the above mentioned technologies by virtue of covalently attaching abacavir sulfate to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absorption by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising abacavir microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and abacavir sulfate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW001P

abacavir sulfate preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting abacavir sulfate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering abacavir sulfate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, abacavir sulfate is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, abacavir sulfate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and abacavir sulfate is released from the composition by dissolution of the microencapsulating agent. In another preferred CW001P

embodiment, abacavir sulfate is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, abacavir sulfate is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching abacavir sulfate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, abacavir sulfate and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW001P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize abacavir sulfate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of abacavir sulfate. Furthermore, active agents can be combined to produce synergistic effects.

Also, absorption of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Abacavir sulfate is the subject of U.S. Patent Numbers 5,034,394 and 5,089,500, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises abacavir sulfate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary structure and the spatial arrangement of the side chains constitute the tertiary structure. CW001P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of irreversible chemical or conformation processes. CW001P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are preferred. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW001P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absorption is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absorption can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Bg (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's CW001P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active CW001P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, abacavir sulfate is covalently attached to the polypeptide via its alcohol group or, alternatively, its amino group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. CW001P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absorption of the peptides.

Preferably, the resultant peptide-abacavir sulfate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed. CW001P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- CW001P

hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terniinus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, CW001P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

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What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and abacavir sulfate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein abacavir sulfate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant. CW001P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein abacavir sulfate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing abacavir sulfate from said composition in a pH-dependent manner.

19. A method for protecting abacavir sulfate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of abacavir sulfate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching abacavir sulfate to said polypeptide.

21. A method for delivering abacavir sulfate to a patient comprising administering to said patient a composition comprising: a polypeptide; and abacavir sulfate covalently attached to said polypeptide.

22. The method of claim 21 wherein abacavir sulfate is released from said composition by an enzyme-catalyzed release. CW001P

23. The method of claim 21 wherein abacavir sulfate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and abacavir sulfate covalently attached to the polypeptide. Also provided is a method for delivery of abacavir sulfate to a patient comprising administering to the patient a composition comprising a polypeptide and abacavir sulfate covalently attached to the polypeptide. Also provided is a method for protecting abacavir sulfate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of abacavir sulfate from a composition comprising covalently attaching it to a polypeptide.

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A NOVtøL PHARMACEUTICAL COMPOUND CONTAINING ABARELIX AND METHODS OF MAKING AND USING SAME

FTELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to abarelix, as well as methods for protecting and administering abarelix. This novel compound, referred to as a CARRffiRWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Abarelix is a known pharmaceutical agent that is used in the treatment of prostate cancer, acting as a gonadotropin-releasing hormone antagonist. Its chemical name is N- acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L- seryl-N-methyl-L-tyrosyl-D-asparagynyl-L-N6-(l-methylethyl)-L-lysyl-L-prolyl-D- alaninamide. Abarelix is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is as follows:

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The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absorbtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absorption of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release CW002P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drug conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW002P

linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absorption through the brash-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (abarelix) ' to a polymer of peptides or amino acids. The invention is distinguished from the above mentioned technologies by virtue of covalently attaching abarelix to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absorption by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising abarelix microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and abarelix covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, CW002P

(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Abarelix preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting abarelix from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering abarelix to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the CW002P

polypeptide. In a preferred embodiment, abarelix is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, abarelix is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and abarelix is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, abarelix is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, abarelix is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching abarelix to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, abarelix and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a CW002P

carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize abarelix and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of abarelix. Furthermore, active agents can be combined to produce synergistic effects. Also, absorption of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises abarelix covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary structure and the spatial arrangement of the side chains constitute the tertiary structure. CW002P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of irreversible chemical or conformation processes. CW002P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are preferred. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW002P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absorption is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absorption can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW002P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW002P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, abarelix is covalently attached to the polypeptide via the free alcohol group or, alternatively, through one of its amino groups.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW002P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absorption of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absorption of the peptides.

Preferably, the resultant peptide-abarelix conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW002P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AciαVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW002P

hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW002P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW002P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and abarelix covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein abarelix is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW002P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein abarelix is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing abarelix from said composition in a pH-dependent manner.

19. A method for protecting abarelix from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of abarelix from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching abarelix to said polypeptide.

21. A method for delivering abarelix to a patient comprising administering to said patient a composition comprising: a polypeptide; and abarelix covalently attached to said polypeptide.

22. The method of claim 21 wherein abarelix is released from said composition by an enzyme-catalyzed release.

18 CW002P

23. The method of claim 21 wherein abarelix is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and abarelix covalently attached to the polypeptide. Also provided is a method for delivery of abarelix to a patient comprising administering to the patient a composition comprising a polypeptide and abarelix covalently attached to the polypeptide. Also provided is a method for protecting abarelix from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling- release of abarelix from a composition comprising covalently attaching it to the polypeptide.

19 CW003P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ACARBOSE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to acarbose, as well as methods for protecting and administering acarbose. This novel compound, referred to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Acarbose is a known pharmaceutical agent that is used in the treatment of type II diabetes. Its chemical name is O-4,6-dideoxy-4-[[[lS-(lalpha,4alpha,5beta,6alpha)]- 4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-l-yl]amino]-alpha-D-glucopyranosyl- (l-4)-O-alpha-D-glucopyranosyl-(l-4)-D-glucose. Its structure is as follows:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absorbtion; targeted delivery to particular tissue/cell type; CW003P

and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absorption of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations. CW003P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW003P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absorption through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (acarbose) to a polymer of peptides or amino acids. The invention is distinguished from the above mentioned technologies by virtue of covalently attaching acarbose to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absorption by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising acarbose microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and acarbose covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW003P

heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

acarbose preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting acarbose from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering acarbose to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, acarbose is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, acarbose is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. CW003P

In another preferred embodiment, the composition further comprises a microencapsulating agent and acarbose is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, acarbose is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, acarbose is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching acarbose to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, acarbose and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW003P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize acarbose and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of acarbose. Furthermore, active agents can be combined to produce synergistic effects. Also, absorption of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Acarbose is the subject of U.S. Patent Number 4,904,769, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises acarbose covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary structure and the spatial arrangement of the side chains constitute the tertiary structure. CW003P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil^' drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of irreversible chemical or conformation processes. CW003P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are preferred. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW003P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW003P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW003P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, acarbose is covalently attached to the polypeptide via any of the free hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW003P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-acarbose conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW003P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AciαVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Anύne/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW003P

hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylarninopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW003P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW003P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and acarbose covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein acarbose is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW003P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein acarbose is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing acarbose from said composition in a pH-dependent manner.

19. A method for protecting acarbose from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of acarbose from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching acarbose to said polypeptide.

21. A method for delivering acarbose to a patient comprising administering to said patient a composition comprising: a polypeptide; and acarbose covalently attached to said polypeptide.

22. The method of claim 21 wherein acarbose is released from said composition by an enzyme-catalyzed release.

18 CW003P

23. The method of claim 21 wherein acarbose is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and acarbose covalently attached to the polypeptide. Also provided is a method for delivery of acarbose to a patient comprising administering to the patient a composition comprising a polypeptide and acarbose covalently attached to the polypeptide. Also provided is a method for protecting acarbose from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of acarbose from a composition comprising covalently attaching it to the polypeptide.

19 CW004P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ACETAMINOPHEN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to acetaminophen, as well as methods for protecting and administering acetaminophen. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Acetaminophen is a known pharmaceutical agent that is used in the treatment of minor aches and pains. Its chemical name is N-acetyl-p-aminophenol. Acetaminophen is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absorbtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW004P

invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW004P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drug conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW004P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (acetaminophen) to a polymer of peptides or amino acids. The invention is distinguished from the above mentioned technologies by virtue of covalently attaching acetaminophen to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising acetaminophen microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and acetaminophen covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

acetaminophen preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide. CW004P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting acetaminophen from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering acetaminophen to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, acetaminophen is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, acetaminophen is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and acetaminophen is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, acetaminophen is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, acetaminophen is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW004P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching acetaminophen to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, acetaminophen and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize acetaminophen and prevent its digestion in the stomach. In CW004P

addition, the pharmacologic effect can be prolonged by delayed release of acetaminophen. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises acetaminophen covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary structure and the spatial arrangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW004P

and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of irreversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will CW004P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are preferred. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the CW004P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order

10 CW004P

to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

1 1 CW004P

preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, acetaminophen is covalently attached to the polypeptide via its hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

12 CW004P

intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-acetaminophen conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

13 CW004P

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

14 CW004P

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW004P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and acetaminophen covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

,8. The composition of claim 1 wherein acetaminophen is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW004P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein acetaminophen is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing acetaminophen from said composition in a pH-dependent manner.

19. A method for protecting acetaminophen from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of acetaminophen from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching acetaminophen to said polypeptide.

21. A method for delivering acetaminophen to a patient comprising administering to said patient a composition comprising: a polypeptide; and acetaminophen covalently attached to said polypeptide.

22. The method of claim 21 wherein acetaminophen is released from said composition by an enzyme-catalyzed release.

17 CW004P

23. The method of claim 21 wherein acetaminophen is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW005P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ACETAMINOPHEN AND CODEINE AND METHODS OF MAKING AND

USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to both acetaminophen and codeine, as well as methods for protecting and administering acetaminophen and codeine together. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Acetaminophen is a known pharmaceutical agent that is used in the treatment of minor aches and pains. Its chemical name is N-acetyl-p-aminophenol. It is often used in combination with codeine, whose chemical name is 7,8-didehydro-4,5-α-epoxy-3- methoxy-17-methylmephorninan-6α-ol. Both are commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absorbtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken CW005P

under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent CW005P

in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR CW005P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (acetaminophen and codeine) to a polymer of peptides or amino acids. The invention is distinguished from the above mentioned technologies by virtue of covalently attaching acetaminophen and codeine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising acetaminophen and codeine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and acetaminophen and codeine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

acetaminophen and codeine preferably is covalently attached to a side chain, the

N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached CW005P

to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting acetaminophen and codeine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering acetaminophen and codeine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, acetaminophen and codeine are released from the composition by an enzyme-catalyzed release. In another preferred embodiment, acetaminophen and codeine are released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and acetaminophen and codeine are released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, acetaminophen and codeine are released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, acetaminophen and codeine are released from the composition in a sustained release. In yet another preferred embodiment, the CW005P

composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching acetaminophen to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, acetaminophen and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW005P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize acetaminophen and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of acetaminophen. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises acetaminophen covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary structure and the spatial arrangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW005P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of irreversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW005P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are preferred. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW005P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW005P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW005P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, acetaminophen and codeine are covalently attached to the polypeptide via its hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW005P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-acetaminophen conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW005P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW005P

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW005P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and acetaminophen covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein acetaminophen and codeine are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW005P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein acetaminophen and codeine are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing acetaminophen from said composition in a pH-dependent manner.

19. A method for protecting acetaminophen from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of acetaminophen from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching acetaminophen to said polypeptide.

21. A method for delivering acetaminophen to a patient comprising administering to said patient a composition comprising: a polypeptide; and acetaminophen covalently attached to said polypeptide.

22. The method of claim 21 wherein acetaminophen and codeine are released from said composition by an enzyme-catalyzed release.

17 CW005P

23. The method of claim 21 wherein acetaminophen and codeine are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW005P

Abstract

A composition comprising a polypeptide and acetaminophen covalently attached to the polypeptide. Also provided is a method for delivery of acetaminophen to a patient comprising administering to the patient a composition comprising a polypeptide and acetaminophen covalently attached to the polypeptide. Also provided is a method for protecting acetaminophen from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of acetaminophen from a composition comprising covalently attaching it to the polypeptide.

19 CW006P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING PROPOXYPHENE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to propoxyphene, as well as methods for protecting and administering propoxyphene. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Propoxyphene is a known pharmaceutical agent that is used in the treatment of pain. It is a mild narcotic analgesic. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is as follows:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW006P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW006P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW006P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (propoxyphene) to a polymer of peptides or amino acids. The invention is distinguished from the above mentioned technologies by virtue of covalently attaching propoxyphene to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising propoxyphene microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and propoxyphene covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

propoxyphene preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW006P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting propoxyphene from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering propoxyphene to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, propoxyphene is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, propoxyphene is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and propoxyphene is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, propoxyphene is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, propoxyphene is CW006P

released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching propoxyphene to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, propoxyphene and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW006P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize propoxyphene and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of propoxyphene. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises propoxyphene covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW006P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW006P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW006P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW006P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

T e alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW006P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, propoxyphene is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW006P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-propoxyphene conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW006P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW006P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl GIutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW006P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and propoxyphene covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein propoxyphene is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW006P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein propoxyphene is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing propoxyphene from said composition in a pH-dependent manner.

19. A method for protecting propoxyphene from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of propoxyphene from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching propoxyphene to said polypeptide.

21. A method for delivering propoxyphene to a patient comprising administering to said patient a composition comprising: a polypeptide; and propoxyphene covalently attached to said polypeptide.

22. The method of claim 21 wherein propoxyphene is released from said composition by an enzyme-catalyzed release.

17 CW006P

23. The method of claim 21 wherein propoxyphene is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW006P

Abstract

A composition comprising a polypeptide and propoxyphene covalently attached to the polypeptide. Also provided is a method for delivery of propoxyphene to a patient comprising administering to the patient a composition comprising a polypeptide and propoxyphene covalently attached to the polypeptide. Also provided is a method for protecting propoxyphene from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of propoxyphene from a composition comprising covalently attaching it to the polypeptide.

19 CW007P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ACETYLSALICYLIC ACID AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to acetylsalicylic acid, as well as methods for protecting and administering acetylsalicylic acid. This novel compound, refeoed to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Acetylsalicylic acid is a known pharmaceutical agent that is used in the treatment of minor aches and pains. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absorbtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW007P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW007P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drug conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW007P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (acetylsalicylic acid) to a polymer of peptides or amino acids. The invention is distinguished from the above mentioned technologies by virtue of covalently attaching acetylsalicylic acid to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising acetylsalicylic acid microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and acetylsalicylic acid covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

acetylsalicylic acid preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide. CW007P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting acetylsalicylic acid from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering acetylsalicylic acid to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, acetylsalicylic acid is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, acetylsalicylic acid is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and acetylsalicylic acid is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, acetylsalicylic acid is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, acetylsalicylic acid is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW007P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching acetylsalicylic acid to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, acetylsalicylic acid and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize acetylsalicylic acid and prevent its digestion in the stomach. CW007P

In addition, the pharmacologic effect can be prolonged by delayed release of acetylsalicylic acid. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises acetylsalicylic acid covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW007P

and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will CW007P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the CW007P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order

10 CW007P

to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

1 1 CW007P

preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, acetylsalicylic acid is covalently attached to the polypeptide via the hydroxy group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

12 CW007P

intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-acetylsalicylic acid conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

13 CW007P

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

14 CW007P

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW007P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and acetylsalicylic acid covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein acetylsalicylic acid is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW007P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein acetylsalicylic acid is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing acetylsalicylic acid from said composition in a pH-dependent manner.

19. A method for protecting acetylsalicylic acid from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of acetylsalicylic acid from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching acetylsalicylic acid to said polypeptide.

21. A method for delivering acetylsalicylic acid to a patient comprising administering to said patient a composition comprising: a polypeptide; and acetylsalicylic acid covalently attached to said polypeptide.

22. The method of claim 21 wherein acetylsalicylic acid is released from said composition by an enzyme-catalyzed release.

17 CW007P

23. The method of claim 21 wherein acetylsalicylic acid is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW007P

Abstract

A composition comprising a polypeptide and acetylsalicylic acid covalently attached to the polypeptide. Also provided is a method for delivery of acetylsalicylic acid to a patient comprising administering to the patient a composition comprising a polypeptide and acetylsalicylic acid covalently attached to the polypeptide. Also provided is a method for protecting acetylsalicylic acid from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of acetylsalicylic acid from a composition comprising covalently attaching it to the polypeptide.

19 CW008P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ACITRETIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to acitretin, as well as methods for protecting and administering acitretin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Acitretin is a known pharmaceutical agent that is used in the treatment of psoriasis. Its chemical name is (all-E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7- dimethyl-2,4,6,8-nonatetraenoic acid. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW008P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the, active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW008P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW008P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (acitretin) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching acitretin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising acitretin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and acitretin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Acitretin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW008P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teπninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting acitretin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering acitretin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, acitretin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, acitretin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and acitretin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, acitretin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, acitretin is released from the composition in a sustained release. CW008P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching acitretin to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, acitretin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW008P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize acitretin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of acitretin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises acitretin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW008P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW008P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW008P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW008P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW008P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, acitretin is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW008P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-acitretin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW008P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW008P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW008P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and acitretin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein acitretin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW008P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein acitretin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing acitretin from said composition in a pH-dependent manner.

19. A method for protecting acitretin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of acitretin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching acitretin to said polypeptide.

21. A method for delivering acitretin to a patient comprising administering to said patient a composition comprising: a polypeptide; and acitretin covalently attached to said polypeptide.

22. The method of claim 21 wherein acitretin is released from said composition by an enzyme-catalyzed release.

17 CW008P

23. The method of claim 21 wherein acitretin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW008P

Abstract

A composition comprising a polypeptide and acitretin covalently attached to the polypeptide. Also provided is a method for delivery of acitretin to a patient comprising administering to the patient a composition comprising a polypeptide and acitretin covalently attached to the polypeptide. Also provided is a method for protecting acitretin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of acitretin from a composition comprising covalently attaching it to the polypeptide.

19 CW009P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ACTIVATED PROTEIN C AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to activated protein C, as well as methods for protecting and administering activated protein C. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Activated protein C is a known pharmaceutical agent that is used in the treatment of blood clots. Its stracture is well known and it is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW009P

invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW009P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW009P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (activated protein C) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching activated protein C to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising activated protein C microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and activated protein C covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Activated protein C preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide. CW009P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting activated protein C from degradation comprising covalently attaching it to a polypeptide. i The invention also provides a method for delivering activated protein C to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, activated protein C is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, activated protein C is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and activated protein C is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, activated protein C is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, activated protein C is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW009P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching activated protein C to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, activated protein C and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize activated protein C and prevent its digestion in the stomach. CW009P

In addition, the pharmacologic effect can be prolonged by delayed release of activated protein C. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises activated protein C covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW009P

and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will CW009P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the CW009P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order

10 CW009P

to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroforrnate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

1 1 CW009P

preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, activated protein C is covalently attached to the polypeptide via a peptide bond.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

12 CW009P

intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-activated protein C conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW009P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW009P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW009P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and activated protein C covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein activated protein C is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

16 CW009P

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein activated protein C is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing activated protein C from said composition in a pH-dependent manner.

19. A method for protecting activated protein C from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of activated protein C from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching activated protein C to said polypeptide.

21. A method for delivering activated protein C to a patient comprising administering to said patient a composition comprising:

17 CW009P

a polypeptide; and activated protein C covalentiy attached to said polypeptide.

22. The method of claim 21 wherein activated protein C is released from said composition by an enzyme-catalyzed release.

23. The method of claim 21 wherein activated protein C is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW009P

Abstract

A composition comprising a polypeptide and activated protein C covalently attached to the polypeptide. Also provided is a method for delivery of activated protein C to a patient comprising administering to the patient a composition comprising a polypeptide and activated protein C covalently attached to the polypeptide. Also provided is a method for protecting activated protein C from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of activated protein C from a composition comprising covalently attaching it to the polypeptide.

19 CW010P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ACYCLOVIR AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to acyclovir, as well as methods for protecting and administering acyclovir. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Acyclovir is a known pharmaceutical agent that is an antiviral drug used in the treatment of herpes simplex viruses. Acyclovir is both commercially available and readily manufactured using public synthetic schemes by those of ordinary skill in the art. Its chemical name is 2-amino-l,9-dihydro-9-[(2-hydroxyethoxy)methyl]-6H-purin-6-one. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; CW010P

and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations. CW010P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW010P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (acyclovir) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching acyclovir to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising acyclovir microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and acyclovir covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW010P

heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Acyclovir preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting acyclovir from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering acyclovir to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, acyclovir is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, acyclovir is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. CW010P

In another prefeoed embodiment, the composition further comprises a microencapsulating agent and acyclovir is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, acyclovir is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, acyclovir is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching acyclovir to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, acyclovir and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW010P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize acyclovir and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of acyclovir. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises acyclovir covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW010P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW010P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW010P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW010P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW010P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, acyclovir is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW010P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-acyclovir conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW010P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW010P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW010P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and acyclovir covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein acyclovir is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW010P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein acyclovir is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing acyclovir from said composition in a pH-dependent manner.

19. A method for protecting acyclovir from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of acyclovir from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching acyclovir to said polypeptide.

21. A method for delivering acyclovir to a patient comprising administering to said patient a composition comprising: a polypeptide; and acyclovir covalently attached to said polypeptide.

22. The method of claim 21 wherein acyclovir is released from said composition by an enzyme-catalyzed release.

17 CW010P

23. The method of claim 21 wherein acyclovir is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW010P

Abstract

A composition comprising a polypeptide and acyclovir covalently attached to the polypeptide. Also provided is a method for delivery of acyclovir to a patient comprising administering to the patient a composition comprising a polypeptide and acyclovir covalently attached to the polypeptide. Also provided is a method for protecting acyclovir from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of acyclovir from a composition comprising covalently attaching it to the polypeptide.

19 CW011P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ADEFOVIR DIPD7OXIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to adefovir dipivoxil, as well as methods for protecting and administering adefovir dipivoxil. This novel compound, refeoed to as a CARREERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Adefovir dipivoxil is a known pharmaceutical agent that is used in the treatment of ADDS. Its chemical name is [[[2-(6-amino-9H-purin-9-yl)ethoxy]methyl] phosphinylidene]bis(oxymethylene)-2,2-dimethylpropanoic acid. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW011P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique, increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW011P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW011P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (adefovir dipivoxil) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching adefovir dipivoxil to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising adefovir dipivoxil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and adefovir dipivoxil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids. CW011P

Adefovir dipivoxil preferably is covalently attached to a side chain, the N- teoninus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting adefovir dipivoxil from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering adefovir dipivoxil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, adefovir dipivoxil is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, adefovir dipivoxil is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and adefovir dipivoxil is released from the composition by dissolution of the microencapsulating agent. In another prefeoed CW011P

embodiment, adefovir dipivoxil is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, adefovir dipivoxil is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching adefovir dipivoxil to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, adefovir dipivoxil and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW011P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize adefovir dipivoxil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of adefovir dipivoxil. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Adefovir dipivoxil is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The composition of the invention comprises adefovir dipivoxil covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture. CW011P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW011P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW011P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW011P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teoninus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW011P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, adefovir dipivoxil is covalently attached to the polypeptide via the amino group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW011P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-adefovir dipivoxil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW011P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW011P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alky I Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW011P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW011P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and adefovir dipivoxil covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein adefovir dipivoxil is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW011P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein adefovir dipivoxil is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing adefovir dipivoxil from said composition in a pH-dependent manner.

19. A method for protecting adefovir dipivoxil from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of adefovir dipivoxil from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching adefovir dipivoxil to said polypeptide.

21. A method for delivering adefovir dipivoxil to a patient comprising administering to said patient a composition comprising: a polypeptide; and adefovir dipivoxil covalently attached to said polypeptide.

22. The method of claim 21 wherein adefovir dipivoxil is released from said composition by an enzyme-catalyzed release.

18 CW011P

23. The method of claim 21 wherein adefovir dipivoxil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and adefovir dipivoxil covalently attached to the polypeptide. Also provided is a method for delivery of adefovir dipivoxil to a patient comprising administering to the patient a composition comprising a polypeptide and adefovir dipivoxil covalently attached to the polypeptide. Also provided is a method for protecting adefovir dipivoxil from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of adefovir dipivoxil from a composition comprising covalently attaching it to the polypeptide.

19 CW012P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ADENOSINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to adenosine, as well as methods for protecting and administering adenosine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Adenosine is a known pharmaceutical agent that is used as a coronary vasodilator. Its chemical name is 9-alpha-D-ribofuranosyl-9H-purin-6-amine. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW012P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW012P

reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drug conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW012P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (adenosine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching adenosine to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising adenosine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and adenosine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Adenosine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW012P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting adenosine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering adenosine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, adenosine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, adenosine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and adenosine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, adenosine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, adenosine is released from the composition in a sustained release. CW012P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching adenosine to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, adenosine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW012P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize adenosine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of adenosine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Adenosine is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The composition of the invention comprises adenosine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW012P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW012P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW012P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW012P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW012P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, adenosine is covalently attached to the polypeptide via the ribose hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW012P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-adenosine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW012P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW012P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl GIutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW012P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and adenosine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein adenosine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW012P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein adenosine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing adenosine from said composition in a pH-dependent manner.

19. A method for protecting adenosine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of adenosine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching adenosine to said polypeptide.

21. A method for delivering adenosine to a patient comprising administering to said patient a composition comprising: a polypeptide; and adenosine covalently attached to said polypeptide.

22. The method of claim 21 wherein adenosine is released from said composition by an enzyme-catalyzed release.

17 CW012P

23. The method of claim 21 wherein adenosine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW012P

Abstract

A composition comprising a polypeptide and adenosine covalently attached to the polypeptide. Also provided is a method for delivery of adenosine to a patient comprising administering to the patient a composition comprising a polypeptide and adenosine covalently attached to the polypeptide. Also provided is a method for protecting adenosine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of adenosine from a composition comprising covalentiy attaching it to the polypeptide.

19 CW013P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ADRENOCORTICOTROPIC HORMONE AND METHODS OF MAKING AND

USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to adrenocorticotropic hormone, as well as methods for protecting and administering adrenocorticotropic hormone. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Adrenocorticotropic hormone is a known phaonaceutical agent that is useful for the diagnosis of Addison's disease and other conditions in which the functionality of the adrenal cortex is to be determined. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase CW013P

markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some CW013P

technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR CW013P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (adrenocorticotropic hormone) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching adrenocorticotropic hormone to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising adrenocorticotropic hormone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and adrenocorticotropic hormone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Adrenocorticotropic hormone preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is CW013P

covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teπninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting adrenocorticotropic hormone from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering adrenocorticotropic hormone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, adrenocorticotropic hormone is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, adrenocorticotropic hormone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and adrenocorticotropic hormone is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, adrenocorticotropic hormone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, adrenocorticotropic hormone is released from the composition in a sustained release. In yet another prefeoed embodiment, the CW013P

composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching adrenocorticotropic hormone to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, adrenocorticotropic hormone and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW013P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize adrenocorticotropic hormone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of adrenocorticotropic hormone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises adrenocorticotropic hormone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW013P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW013P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of CW013P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW013P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW013P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, adrenocorticotropic hormone is covalently attached to the polypeptide via an amide bond.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW013P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-adrenocorticotropic hormone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW013P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW013P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW013P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW013P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and adrenocorticotropic hormone covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein adrenocorticotropic hormone is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW013P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein adrenocorticotropic hormone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing adrenocorticotropic hormone from said composition in a pH-dependent manner.

19. A method for protecting adrenocorticotropic hormone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of adrenocorticotropic hormone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching adrenocorticotropic hormone to said polypeptide.

21. A method for delivering adrenocorticotropic hormone to a patient comprising administering to said patient a composition comprising: a polypeptide; and adrenocorticotropic hormone covalently attached to said polypeptide.

22. The method of claim 21 wherein adrenocorticotropic hormone is released from said composition by an enzyme-catalyzed release.

18 CW013P

23. The method of claim 21 wherein adrenocorticotropic hormone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and adrenocorticotropic hormone covalently attached to the polypeptide. Also provided is a method for delivery of adrenocorticotropic hormone to a patient comprising administering to the patient a composition comprising a polypeptide and adrenocorticotropic hormone covalently attached to the polypeptide. Also provided is a method for protecting adrenocorticotropic hormone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of adrenocorticotropic hormone from a composition comprising covalently attaching it to the polypeptide.

19 CW014P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALBUTEROL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to albuterol, as well as methods for protecting and administering albuterol. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Albuterol is a known pharmaceutical agent that is used for the symptomatic management of bronchospasm in patients with reversible, obstractive airway disease. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW014P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW014P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW014P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (albuterol) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching albuterol to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising albuterol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and albuterol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Albuterol preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW014P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting albuterol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering albuterol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, albuterol is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, albuterol is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and albuterol is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, albuterol is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, albuterol is released from the composition in a sustained release. CW014P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching albuterol to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, albuterol and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW014P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize albuterol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of albuterol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises albuterol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW014P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW014P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW014P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW014P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW014P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, albuterol is covalently attached to the polypeptide via one of the hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW014P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-albuterol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW014P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW014P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW014P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and albuterol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein albuterol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW014P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein albuterol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing albuterol from said composition in a pH-dependent manner.

19. A method for protecting albuterol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of albuterol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching albuterol to said polypeptide.

21. A method for delivering albuterol to a patient comprising administering to said patient a composition comprising: a polypeptide; and albuterol covalently attached to said polypeptide.

22. The method of claim 21 wherein albuterol is released from said composition by an enzyme-catalyzed release.

17 CW014P

23. The method of claim 21 wherein albuterol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW014P

Abstract

A composition comprising a polypeptide and albuterol covalently attached to the polypeptide. Also provided is a method for delivery of albuterol to a patient comprising administering to the patient a composition comprising a polypeptide and albuterol covalently attached to the polypeptide. Also provided is a method for protecting albuterol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of albuterol from a composition comprising covalently attaching it to the polypeptide.

19 CW015P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALENDRONATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to alendronate, as well as methods for protecting and administering alendronate. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Alendronate is a known pharmaceutical agent that is used for controlling osteoporosis in men. Its chemical name is (4-amino-l-hydroxybutylidene)bisphosphonic acid. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW015P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet fo nulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW015P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW015P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(alendronate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching alendronate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising alendronate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and alendronate covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Alendronate preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW015P

the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting alendronate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering alendronate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, alendronate is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, alendronate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and alendronate is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, alendronate is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, alendronate is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and CW015P

release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching alendronate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, alendronate and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW015P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize alendronate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of alendronate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Alendronate is the subject of U.S. Patent Numbers 4,621,077, 5,358,941, 5,681,950, 5,804,570, 5,849,726, 6,008,207, and 6,090,410, herein incoφorated by reference, which describe how to make that drag.

The composition of the invention comprises alendronate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the CW015P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW015P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW015P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW015P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW015P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, alendronate is covalently attached to the polypeptide via the hydroxyl or phosphate groups.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW015P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-alendronate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW015P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to ■ 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW015P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW015P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and alendronate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein alendronate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW015P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein alendronate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing alendronate from said composition in a pH-dependent manner.

19. A method for protecting alendronate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of alendronate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching alendronate to said polypeptide.

21. A method for delivering alendronate to a patient comprising administering to said patient a composition comprising: a polypeptide; and alendronate covalently attached to said polypeptide.

22. The method of claim 21 wherein alendronate is released from said composition by an enzyme-catalyzed release.

17 CW015P

23. The method of claim 21 wherein alendronate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW015P

Abstract

A composition comprising a polypeptide and alendronate covalently attached to the polypeptide. Also provided is a method for delivery of alendronate to a patient comprising administering to the patient a composition comprising a polypeptide and alendronate covalently attached to the polypeptide. Also provided is a method for protecting alendronate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of alendronate from a composition comprising covalently attaching it to the polypeptide.

19 CW016P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALLOPURINAL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to allopurinal, as well as methods for protecting and administering allopurinal. This novel compound, refeoed to as a CARRDERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Allopurinal is a known pharmaceutical agent that is is a xanthine oxidase inhibitor used in the treatment of gout and selected hyperaricemias. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

CW016P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW016P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW016P

linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brash-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present i vention provides covalent attachment of the active agent (allopurinal) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching allopurinal to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising allopurinal microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and allopurinal covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, CW016P

(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Allopurinal preferably is covalentiy attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting allopurinal from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering allopurinal to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the CW016P

polypeptide. In a prefeoed embodiment, allopurinal is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, allopurinal is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and allopurinal is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, allopurinal is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, allopurinal is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching allopurinal to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, allopurinal and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a CW016P

carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize allopurinal and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of allopurinal. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises allopurinal covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture. CW016P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW016P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW016P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dexfran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW016P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW016P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, allopurinal is covalently attached to the polypeptide via its -NH group.

The polypeptide caoier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW016P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-allopurinal conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW016P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW016P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW016P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW016P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and allopurinal covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein allopurinal is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW016P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein allopurinal is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing allopurinal from said composition in a pH-dependent manner.

19. A method for protecting allopurinal from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of allopurinal from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching allopurinal to said polypeptide.

21. A method for delivering allopurinal to a patient comprising administering to said patient a composition comprising: a polypeptide; and allopurinal covalently attached to said polypeptide.

22. The method of claim 21 wherein allopurinal is released from said composition by an enzyme-catalyzed release.

18 CW016P

23. The method of claim 21 wherein allopurinal is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and allopurinal covalently attached to the polypeptide. Also provided is a method for delivery of allopurinal to a patient comprising administering to the patient a composition comprising a polypeptide and allopurinal covalently attached to the polypeptide. Also provided is a method for protecting allopurinal from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of allopurinal from a composition comprising covalently attaching it to the polypeptide.

19 CW017P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALPHA 1 PROTEINASE INHIBITOR AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to Alpha 1 proteinase inhibitor, as well as methods for protecting and administering Alpha 1 proteinase inhibitor. This novel compound, refeoed to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Alpha 1 proteinase inhibitor is a known pharmaceutical agent that is used in the treatment of emphysema. It is a natural product isolated from human blood, using methods known to those of ordinary skill in the art.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique, increasing the stability of the active agent, such as prolonging shelf CW017P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings, incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW017P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW017P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (Alpha 1 proteinase inhibitor) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching Alpha 1 proteinase inhibitor to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising Alpha 1 proteinase inhibitor microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and Alpha 1 proteinase inhibitor covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Alpha 1 proteinase inhibitor preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet CW017P

another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting Alpha 1 proteinase inhibitor from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering Alpha 1 proteinase inhibitor to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, Alpha 1 proteinase inhibitor is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, Alpha 1 proteinase inhibitor is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and Alpha 1 proteinase inhibitor is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, Alpha 1 proteinase inhibitor is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, Alpha 1 proteinase inhibitor is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The CW017P

adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching Alpha 1 proteinase inhibitor to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, Alpha 1 proteinase inhibitor and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW017P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize Alpha 1 proteinase inhibitor and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of Alpha 1 proteinase inhibitor. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises Alpha 1 proteinase inhibitor covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW017P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW017P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW017P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW017P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW017P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW017P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-Alpha 1 proteinase inhibitor conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be

13 CW017P

stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW017P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alk l Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AlkyI Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW017P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and Alpha 1 proteinase inhibitor covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein Alpha 1 proteinase inhibitor is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW017P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein Alpha 1 proteinase inhibitor is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing Alpha 1 proteinase inhibitor from said composition in a pH-dependent manner.

19. A method for protecting Alpha 1 proteinase inhibitor from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of Alpha 1 proteinase inhibitor from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching Alpha 1 proteinase inhibitor to said polypeptide.

21. A method for delivering Alpha 1 proteinase inhibitor to a patient comprising administering to said patient a composition comprising: a polypeptide; and Alpha 1 proteinase inhibitor covalently attached to said polypeptide.

22. The method of claim 21 wherein Alpha 1 proteinase inhibitor is released from said composition by an enzyme-catalyzed release.

17 CW017P

23. The method of claim 21 wherein Alpha 1 proteinase inhibitor is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW017P

Abstract

A composition comprising a polypeptide and Alpha 1 proteinase inhibitor covalently attached to the polypeptide. Also provided is a method for delivery of Alpha 1 proteinase inhibitor to a patient comprising administering to the patient a composition comprising a polypeptide and Alpha 1 proteinase inhibitor covalently attached to the polypeptide. Also provided is a method for protecting Alpha 1 proteinase inhibitor from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of Alpha 1 proteinase inhibitor from a composition comprising covalently attaching it to the polypeptide.

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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALPRAZALOM AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to alprazalom, as well as methods for protecting and administering alprazalom. This novel compound, refeoed to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its effectiveness.

BACKGROUND OF THE INVENTION

Alprazalom is a known pharmaceutical agent that is used in the treatment of anxiety disorders. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

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The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stabiUty of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW018P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW018P

linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (alprazalom) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching alprazalom to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising alprazalom microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and alprazalom covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, CW018P

(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Alprazalom preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting alprazalom from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering alprazalom to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the CW018P

polypeptide. In a prefeoed embodiment, alprazalom is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, alprazalom is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and alprazalom is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, alprazalom is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, alprazalom is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching alprazalom to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, alprazalom and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side CW018P

chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize alprazalom and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of alprazalom. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

In the present invention, alprazalom is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The composition of the invention comprises alprazalom covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the CW018P

local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the CW018P

amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired. CW018P

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border " membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

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TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For exam iple, a decamer of glycine (MW=588) linked to aspirin would have a total molecular w -e < ig _.ht of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

1 1 CW018P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

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The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, alprazalom is covalently attached to the polypeptide via the zzzzzzz.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

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mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-alprazalom conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

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In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

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Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AlkyI Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

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What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and alprazalom covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein alprazalom is covalently attached to a side chain, the N-terminus or the C-teoninus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein alprazalom is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing alprazalom from said composition in a pH-dependent manner.

19. A method for protecting alprazalom from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of alprazalom from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching alprazalom to said polypeptide.

21. A method for delivering alprazalom to a patient comprising administering to said patient a composition comprising: a polypeptide; and alprazalom covalently attached to said polypeptide.

22. The method of claim 21 wherein alprazalom is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein alprazalom is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and alprazalom covalentiy attached to the polypeptide. Also provided is a method for delivery of alprazalom to a patient comprising administering to the patient a composition comprising a polypeptide and alprazalom covalently attached to the polypeptide. Also provided is a method for protecting alprazalom from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of alprazalom from a composition comprising covalently attaching it to the polypeptide.

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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALPROSTADIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to alprostadil, as weU as methods for protecting and administering alprostadil. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Alprostadil is a known pharmaceutical agent that is used in the treatment of male erectile dysfunction. Its chemical name is ( 11 alpha, 13E, 15S)- 11 , 15-dihydroxy-9- oxoprost-13-en-l-oic acid. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active phaonaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW019P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW019P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW019P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(alprostadil) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching alprostadil to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising alprostadil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and alprostadil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Alprostadil preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW019P

the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting alprostadil from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering alprostadil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, alprostadil is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, alprostadil is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and alprostadil is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, alprostadil is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, alprostadil is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW019P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching alprostadil to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, alprostadil and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW019P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize alprostadil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of alprostadil. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Alprostadil is the subject of U.S. Patent Number 5,741,523, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises alprostadil covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW019P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW019P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW019P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW019P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW019P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, alprostadil is covalently attached to the polypeptide via the carboxylic acid.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW019P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-alprostadil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW019P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW019P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW019P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and alprostadil covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein alprostadil is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW019P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein alprostadil is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing alprostadil from said composition in a pH-dependent manner.

19. A method for protecting alprostadil from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of alprostadil from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching alprostadil to said polypeptide.

21. A method for delivering alprostadil to a patient comprising administering to said patient a composition comprising: a polypeptide; and alprostadil covalently attached to said polypeptide.

22. The method of claim 21 wherein alprostadil is released from said composition by an enzyme-catalyzed release.

17 CW019P

23. The method of claim 21 wherein alprostadil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW019P

Abstract

A composition comprising a polypeptide and alprostadil covalently attached to the polypeptide. Also provided is a method for delivery of alprostadil to a patient comprising administering to the patient a composition comprising a polypeptide and alprostadil covalentiy attached to the polypeptide. Also provided is a method for protecting alprostadil from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of alprostadil from a composition comprising covalentiy attaching it to the polypeptide.

19 CW020P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALTINICLINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to altinicline, as well as methods for protecting and administering altinicline. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pha naceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Altinicline is a known pharmaceutical agent that is used in the treatment of Parkinson's disease. Its chemical name is 3-ethynyl-5-[(2S)-l-methyl-2- pyoolidinyljpyridine. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active phaonaceutical agent, an adjuvant, or an inhibitor. CW020P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW020P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drug conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW020P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (altinicline) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching altinicline to the N- terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising altinicline microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and altinicline covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Altinicline preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW020P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting altinicline from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering altinicline to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, altinicline is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, altinicline is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and altinicline is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, altinicline is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, altinicline is released from the composition in a sustained release. CW020P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching altinicline to a side chain of an amino acid to form an active agent amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, altinicline and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW020P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize altinicline and prevent its digestion in the stomach. In addition, the phaonacologic effect can be prolonged by delayed release of altinicline. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Altinicline is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The composition of the invention comprises altinicline covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW020P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW020P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW020P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW020P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW020P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not hmited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, altinicline is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW020P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-altinicline conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW020P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW020P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- lkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW020P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and altinicline covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein altinicline is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW020P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein altinicline is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing altinicline from said composition in a pH-dependent manner.

19. A method for protecting altinicline from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of altinicline from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching altinicline to said polypeptide.

21. A method for delivering altinicline to a patient comprising administering to said patient a composition comprising: a polypeptide; and altinicline covalently attached to said polypeptide.

22. The method of claim 21 wherein altinicline is released from said composition by an enzyme-catalyzed release.

17 CW020P

23. The method of claim 21 wherein altinicline is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW020P

Abstract

A composition comprising a polypeptide and altinicline covalently attached to the polypeptide. Also provided is a method for delivery of altinicline to a patient comprising administering to the patient a composition comprising a polypeptide and altinicline covalentiy attached to the polypeptide. Also provided is a method for protecting altinicline from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of altinicline from a composition comprising covalentiy attaching it to the polypeptide.

19 CW021P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AMTFOSTINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to amifostine, as well as methods for protecting and administering amifostine. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Amifostine is a known pharmaceutical agent that is used in the treatment of moderate to severe xerostomia in patients with head and neck cancer undergoing postoperative radiotherapy. Its chemical name is 2-[(3-anτinopropyl)amino]ethanethiol dihydrogen phosphate. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW021P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW021P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW021P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(amifostine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching amifostine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising amifostine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and amifostine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Amifostine preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW021P

the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting amifostine from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering amifostine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, amifostine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, amifostine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and amifostine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, amifostine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, amifostine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW021P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching amifostine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, amifostine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW021P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize amifostine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of amifostine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Amifostine is the subject of U.S. Patent Numbers 5,424,471, 5,591,731, and 5,994,409, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises amifostine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW021P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW021P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW021P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW021P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW021P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, amifostine is covalently attached to the polypeptide via the phosphate group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW021P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-amifostine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW021P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW021P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW021P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and amifostine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein amifostine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW021P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein amifostine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing amifostine from said composition in a pH-dependent manner.

19. A method for protecting amifostine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of amifostine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching amifostine to said polypeptide.

21. A method for delivering amifostine to a patient comprising administering to said patient a composition comprising: a polypeptide; and amifostine covalently attached to said polypeptide.

22. The method of claim 21 wherein amifostine is released from said composition by an enzyme-catalyzed release.

17 CW021P

23. The method of claim 21 wherein amifostine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW021P

Abstract

A composition comprising a polypeptide and amifostine covalently attached to the polypeptide. Also provided is a method for delivery of amifostine to a patient comprising administering to the patient a composition comprising a polypeptide and amifostine covalently attached to the polypeptide. Also provided is a method for protecting amifostine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of amifostine from a composition comprising covalently attaching it to the polypeptide.

19 CW022P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AMIODARONE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to amiodarone, as well as methods for protecting and administering amiodarone. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Amiodarone is a known pharmaceutical agent that is used in the treatment of cardiac aohythmia. Its chemical name is (2-butyl-3-benzofuranyl)[4-[2-

(diethylamino)ethoxy]-3,5-diiodophenyl]methanone. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW022P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW022P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW022P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (amiodarone) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching amiodarone to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising amiodarone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and amiodarone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW022P

Amiodarone preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting amiodarone from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering amiodarone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, amiodarone is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, amiodarone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and amiodarone is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, CW022P

amiodarone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, amiodarone is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching amiodarone to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, amiodarone and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW022P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize amiodarone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of amiodarone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises amiodarone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW022P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW022P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW022P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW022P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teiminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW022P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, amiodarone is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW022P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-amiodarone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW022P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW022P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW022P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and amiodarone covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein amiodarone is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW022P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein amiodarone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing amiodarone from said composition in a pH-dependent manner.

19. A method for protecting amiodarone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of amiodarone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching amiodarone to said polypeptide.

21. A method for delivering amiodarone to a patient comprising administering to said patient a composition comprising: a polypeptide; and amiodarone covalently attached to said polypeptide.

22. The method of claim 21 wherein amiodarone is released from said composition by an enzyme-catalyzed release.

17 CW022P

23. The method of claim 21 wherein amiodarone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW022P

Abstract

A composition comprising a polypeptide and amiodarone covalently attached to the polypeptide. Also provided is a method for delivery of amiodarone to a patient comprising administering to the patient a composition comprising a polypeptide and amiodarone covalently attached to the polypeptide. Also provided is a method for protecting amiodarone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of amiodarone from a composition comprising covalentiy attaching it to the polypeptide.

19 CW023P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AMITRIPTYLINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to amitriptyline, as well as methods for protecting and administering amitriptyline. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Amitriptyline is a known pharmaceutical agent that is used in the treatment of depression. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability CW023P

of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified CW023P

amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that CW023P

incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR apphcation, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (amitriptyline) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching amitriptyline to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising amitriptyline microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and amitriptyline covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a CW023P

heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Amitriptyline preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting amitriptyline from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering amitriptyline to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, amitriptyline is released from the composition CW023P

by an enzyme-catalyzed release. In another preferred embodiment, amitriptyline is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and amitriptyline is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, amitriptyline is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, amitriptyline is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching amitriptyline to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, amitriptyline and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a CW023P

carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize amitriptyline and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of amitriptyline. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises amitriptyline covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture. CW023P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW023P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW023P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW023P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW023P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, amitriptyline is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

12 CW023P

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

13 CW023P

Preferably, the resultant peptide-amitriptyline conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated

14 CW023P

solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

15 CW023P

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW023P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and amitriptyline covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein amitriptyline is covalentiy attached to a side chain, the N-terminus or the C-teoninus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW023P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein amitriptyline is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing amitriptyline from said composition in a pH-dependent manner.

19. A method for protecting amitriptyline from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of amitriptyline from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching amitriptyline to said polypeptide.

21. A method for delivering amitriptyline to a patient comprising administering to said patient a composition comprising: a polypeptide; and amitriptyline covalently attached to said polypeptide.

22. The method of claim 21 wherein amitriptyline is released from said composition by an enzyme-catalyzed release.

18 CW023P

23. The method of claim 21 wherein amitriptyline is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and amitriptyline covalently attached to the polypeptide. Also provided is a method for delivery of amitriptyline to a patient comprising administering to the patient a composition comprising a polypeptide and amitriptyline covalently attached to the polypeptide. Also provided is a method for protecting amitriptyline from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of amitriptyline from a composition comprising covalently attaching it to the polypeptide.

19 CW024P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AMLODIPINE BES YLATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to amlodipine besylate , as well as methods for protecting and administering amlodipine besylate . This novel compound, refened to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Amlodipine besylate is a known pharmaceutical agent that is used in the treatment and prevention of myocardial infarction and stroke. Its chemical name is 2-[(2- aminoethoxy)methyl]-4-(2-chlorophenyl)-l,4-dihydro-6-methyl-3,5-pyridinedicarboxylic acid, 3-ethyl 5-methyl ester monobenzenesulfonate. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW024P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and- time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW024P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW024P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (amlodipine besylate ) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching amlodipine besylate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising amlodipine besylate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and amlodipine besylate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Amlodipine besylate preferably is covalently attached to a side chain, the N- teoninus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active CW024P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, 'the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting amlodipine besylate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering amlodipine besylate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefened embodiment, amlodipine besylate is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, amlodipine besylate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and amlodipine besylate is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, amlodipine besylate is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, amlodipine CW024P

besylate is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching amlodipine besylate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, amlodipine besylate and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW024P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize amlodipine besylate and prevent its digestion in the stomach. In addition, the phaonacologic effect can be prolonged by delayed release of amlodipine besylate . Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Amlodipine besylate is the subject of U.S. Patent Numbers 4,572,909 and

4,879,303, herein incoφorated by reference, which describe how to make that drug.

The composition of the invention comprises amlodipine besylate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW024P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW024P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW024P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ό (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW024P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus apphcation, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW024P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, amlodipine besylate is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW024P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-amlodipine besylate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW024P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW024P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW024P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and amlodipine besylate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein amlodipine besylate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW024P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein amlodipine besylate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing amlodipine besylate from said composition in a pH-dependent manner.

19. A method for protecting amlodipine besylate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of amlodipine besylate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching amlodipine besylate to said polypeptide.

21. A method for delivering amlodipine besylate to a patient comprising administering to said patient a composition comprising: a polypeptide; and amlodipine besylate covalently attached to said polypeptide.

22. The method of claim 21 wherein amlodipine besylate is released from said composition by an enzyme-catalyzed release.

17 CW024P

23. The method of claim 21 wherein amlodipine besylate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW024P

Abstract

A composition comprising a polypeptide and amlodipine besylate covalently attached to the polypeptide. Also provided is a method for delivery of amlodipine besylate to a patient comprising administering to the patient a composition comprising a polypeptide and amlodipine besylate covalently attached to the polypeptide. Also provided is a method for protecting amlodipine besylate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of amlodipine besylate from a composition comprising covalently attaching it to the polypeptide.

19 CW025P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AMLODIPINE

BESYLATE AND BENAZEPRIL AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to amlodipine besylate, as well as methods for protecting and administering amlodipine besylate and benazepril. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Amlodipine besylate is a known pharmaceutical agent that is used in the treatment and prevention of myocardial infarction and stroke. Its chemical name is 2-[(2- aminoethoxy)methyl]-4-(2-chlorophenyl)-l,4-dihydro-6-methyl-3,5-pyridinedicarboxylic acid, 3-ethyl 5-methyl ester monobenzenesulfonate. Its stracture is:

Benazepril has the chemical name [S-(R*,R*)]-3-[[l-(ethoxycarbonyl)-3- phenylpropyl]amino]-2,3,4,5-tetrahydro-2-oxo- 1 H- 1 -benzazepine- 1 -acetic acid. It is available commercially or can be made by those of skill in the art. Its stracture is as follows: CW025P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach. CW025P

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drug conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where CW025P

the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (amlodipine besylate and benazepril) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching amlodipine besylate and benazepril to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively CW025P

hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising amlodipine besylate and benazepril microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and amlodipine besylate and benazepril covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Amlodipine besylate and benazepril preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In CW025P

another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting amlodipine besylate and benazepril from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering amlodipine besylate and benazepril to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, amlodipine besylate and benazepril are released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, amlodipine besylate and benazepril are released in a time- dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and amlodipine besylate and benazepril are released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, amlodipine besylate and benazepril are released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, amlodipine besylate and benazepril is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching amlodipine besylate and benazepril to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and CW025P

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, amlodipine besylate and benazepril and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize amlodipine besylate and benazepril and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of amlodipine besylate and benazepril. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action. CW025P

Amlodipine besylate is the subject of U.S. Patent Numbers 4,572,909 and 4,879,303, herein incoφorated by reference, which describe how to make that drug.

The composition of the invention comprises amlodipine besylate and benazepril covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with CW025P

protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will CW025P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release p primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the

10 CW025P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order

11 CW025P

to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

12 CW025P

preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, amlodipine besylate is covalently attached to the polypeptide via the amino group. Benazepril is attached via its carboxylic acid.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

13 CW025P

intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-amlodipine besylate and benazepril conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

14 CW025P

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

15 CW025P

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW025P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and amlodipine besylate and benazepril covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein amlodipine besylate and benazepril are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW025P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein amlodipine besylate and benazepril are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing amlodipine besylate and benazepril from said composition in a pH-dependent manner.

19. A method for protecting amlodipine besylate and benazepril from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of amlodipine besylate and benazepril from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching amlodipine besylate and benazepril to said polypeptide.

21. A method for delivering amlodipine besylate and benazepril to a patient comprising administering to said patient a composition comprising: a polypeptide; and amlodipine besylate and benazepril covalently attached to said polypeptide.

22. The method of claim 21 wherein amlodipine besylate and benazepril are released from said composition by an enzyme-catalyzed release.

18 CW025P

23. The method of claim 21 wherein amlodipine besylate and benazepril is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and amlodipine besylate and benazepril covalently attached to the polypeptide. Also provided is a method for delivery of amlodipine besylate and benazepril to a patient comprising administering to the patient a composition comprising a polypeptide and amlodipine besylate and benazepril covalently attached to the polypeptide. Also provided is a method for protecting amlodipine besylate and benazepril from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of amlodipine besylate and benazepril from a composition comprising covalently attaching it to the polypeptide.

19 CW026P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AMOXICILLIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to amoxicillin, as well as methods for protecting and administering amoxicillin. This novel compound, refeoed to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Amoxicillin is a known phaonaceutical agent that is used in the treatment of bacterial infection. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaonaceutical agent, an adjuvant, or an inhibitor. CW026P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings, incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW026P

reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW026P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (amoxicillin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching amoxicillin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising amoxicillin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and amoxicillin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Amoxicillin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW026P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting amoxicillin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering amoxicillin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, amoxicillin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, amoxicillin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and amoxicillin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, amoxicillin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, amoxicillin is released from the composition in a CW026P

sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching amoxicillin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, amoxicillin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is CW026P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize amoxicillin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of amoxicillin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises amoxicillin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW026P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stabihty of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW026P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the canier peptide and the active agent. CW026P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW026P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW026P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, amoxicillin is covalently attached to the polypeptide via the carboxylic acid.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW026P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-amoxicillin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW026P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW026P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alky I Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW026P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and amoxicillin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein amoxicillin is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW026P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein amoxicillin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing amoxicillin from said composition in a pH-dependent manner.

19. A method for protecting amoxicillin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of amoxicillin from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching amoxicillin to said polypeptide.

21. A method for delivering amoxicillin to a patient comprising administering to said patient a composition comprising: a polypeptide; and amoxicillin covalently attached to said polypeptide.

22. The method of claim 21 wherein amoxicillin is released from said composition by an enzyme-catalyzed release.

17 CW026P

23. The method of claim 21 wherein amoxicillin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW026P

Abstract

A composition comprising a polypeptide and amoxicillin covalently attached to the polypeptide. Also provided is a method for delivery of amoxicillin to a patient comprising administering to the patient a composition comprising a polypeptide and amoxicillin covalently attached to the polypeptide. Also provided is a method for protecting amoxicillin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of amoxicillin from a composition comprising covalently attaching it to the polypeptide.

19 CW027P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AMOXICILLIN AND CLAVULANATE AND METHODS OF MAKING AND

USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to amoxicillin and clavulanate, as well as methods for protecting and administering amoxicillin and clavulanate. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Amoxicillin and clavulanate are known pharmaceutical agents used in the treatment of bacterial infections. Each is available commercially and can be made by those of ordinary skill in the art. The structure of amoxicillin is:

The stracture of clavulanate is(Z)-(2Λ,5K )-3-(2-hydroxyethylidene)-7-oxo-4- oxa-l-azabicyclo[3.2.0]-heptane-2-carboxylate.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW027P

compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW027P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW027P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (amoxicillin and clavulanate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching amoxicillin and clavulanate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising amoxicillin and clavulanate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and amoxicillin and clavulanate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW027P

Amoxicillin and clavulanate preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting amoxicillin and clavulanate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering amoxicillin and clavulanate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, amoxicillin and clavulanate is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, amoxicillin and clavulanate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and amoxicillin and clavulanate is released from the composition by dissolution of the CW027P

microencapsulating agent. In another prefeoed embodiment, amoxicillin and clavulanate is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, amoxicillin and clavulanate is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching amoxicillin and clavulanate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, amoxicillin and clavulanate and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW027P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize amoxicillin and clavulanate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of amoxicillin and clavulanate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Amoxicillin and clavulanate is the subject of U.S. Patent Number yyyyy, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises amoxicillin and clavulanate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture. CW027P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stabiUty is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW027P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controUed peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW027P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 ftmprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW027P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teπninus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW027P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, amoxicillin and clavulanate are each covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW027P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophihc compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

13 CW027P

Preferably, the resultant peptide-amoxicillin and clavulanate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

14 CW027P

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically

15 CW027P

overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW027P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and amoxicillin and clavulanate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein amoxicillin and clavulanate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW027P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein amoxicillin and clavulanate is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing amoxicillin and clavulanate from said composition in a pH-dependent manner.

19. A method for protecting amoxicillin and clavulanate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of amoxicillin and clavulanate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching amoxicillin and clavulanate to said polypeptide.

21. A method for delivering amoxicillin and clavulanate to a patient comprising administering to said patient a composition comprising: a polypeptide; and amoxicillin and clavulanate covalently attached to said polypeptide.

22. The method of claim 21 wherein amoxicillin and clavulanate is released from said composition by an enzyme-catalyzed release.

18 CW027P

23. The method of claim 21 wherein amoxicillin and clavulanate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and amoxicillin and clavulanate covalently attached to the polypeptide. Also provided is a method for delivery of amoxicillin and clavulanate to a patient comprising administering to the patient a composition comprising a polypeptide and amoxicillin and clavulanate covalently attached to the polypeptide. Also provided is a method for protecting amoxicilhn and clavulanate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of amoxicillin and clavulanate from a composition comprising covalently attaching it to the polypeptide.

19 CW028P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AMPRENAVIR AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to amprenavir, as well as methods for protecting and administering amprenavir. This novel compound, refeoed to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Amprenavir, a proteinase inhibitor, is a known pharmaceutical agent that is used in the treatment of HIV infection. Its chemical name is [3S-3R*(lR*,2S*)]]-[3-[[(4- aminophenyl)sulfonyl] (2-methylpropyl)amino] -2-hydroxy- 1 - (phenylmethyl)propyl]carbamic acid tetrahydro-3-furanyl ester. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW028P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibUity and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stabihty and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW028P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW028P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is Umited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (amprenavir) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching amprenavir to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising amprenavir microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and amprenavir covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW028P

Amprenavir preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting amprenavir from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering amprenavir to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, amprenavir is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, amprenavir is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and amprenavir is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, amprenavir is CW028P

released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, amprenavir is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching amprenavir to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, amprenavir and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW028P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize amprenavir and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of amprenavir. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Amprenavir is the subject of U.S. Patent Numbers 5,585,397, 5,646,180, and 5,723,490, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises amprenavir covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture. CW028P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oU drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW028P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipopbilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW028P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. GeneraUy, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW028P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oUgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- teiminus of the oUgopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW028P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, amprenavir is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW028P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabUizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epitheha efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-amprenavir conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW028P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW028P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW028P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW028P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and amprenavir covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein amprenavir is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW028P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein amprenavir is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing amprenavir from said composition in a pH-dependent manner.

19. A method for protecting amprenavir from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of amprenavir from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching amprenavir to said polypeptide.

21. A method for delivering amprenavir to a patient comprising administering to said patient a composition comprising: a polypeptide; and amprenavir covalently attached to said polypeptide.

22. The method of claim 21 wherein amprenavir is released from said composition by an enzyme-catalyzed release. CW028P

23. The method of claim 21 wherein amprenavir is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and amprenavir covalently attached to the polypeptide. Also provided is a method for delivery of amprenavir to a patient comprising administering to the patient a composition comprising a polypeptide and amprenavir covalently attached to the polypeptide. Also provided is a method for protecting amprenavir from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of amprenavir from a composition comprising covalently attaching it to the polypeptide.

19 CW029P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ANAGRELIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to anagreUde, as well as methods for protecting and administering anagrelide. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pha naceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Anagrelide is a known pharmaceutical agent that is used as a platelet reducing drag. Its chemical name is 6,7-dichloro-l,5-dihydroimidazo[2,l-b]quinazolin-2(3H)-one. Its structure is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW029P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW029P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW029P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(anagrelide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching anagrelide to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is confroUed, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising anagrelide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and anagrelide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Anagrelide preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW029P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting anagrelide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering anagrelide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, anagrelide is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, anagrelide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and anagrelide is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, anagrelide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, anagrelide is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW029P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching anagrelide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, anagrelide and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW029P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize anagrelide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of anagrelide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Anagrelide is the subject of GB patent 1418822 (1975), (based on U.S. Patent Application Serial Number 223,723, filed in 1972, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises anagrelide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (u) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the CW029P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW029P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophiϋcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW029P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW029P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases. *

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW029P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, anagrelide is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW029P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-anagrelide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW029P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW029P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW029P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and anagrelide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein anagrelide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW029P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein anagrelide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing anagrelide from said composition in a pH-dependent manner.

19. A method for protecting anagrelide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of anagrelide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching anagrelide to said polypeptide.

21. A method for delivering anagrelide to a patient comprising administering to said patient a composition comprising: a polypeptide; and anagrelide covalently attached to said polypeptide.

22. The method of claim 21 wherein anagrelide is released from said composition by an enzyme-catalyzed release.

17 CW029P

23. The method of claim 21 wherein anagrelide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW029P

Abstract

A composition comprising a polypeptide and anagrelide covalently attached to the polypeptide. Also provided is a method for delivery of anagrelide to a patient comprising administering to the patient a composition comprising a polypeptide and anagrelide covalently attached to the polypeptide. Also provided is a method for protecting anagrelide from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of anagrelide from a composition comprising covalently attaching it to the polypeptide.

19 CW030P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ANARITIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to anaritide, as well as methods for protecting and administering anaritide. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Anaritide is a known phaonaceutical agent that is used in the treatment of oliguric acute renal failure. Its chemical name is N-L-arginyl-8-L-methionine-21a-L- phenylalanine-21b-L-arginine-21c-L-tyrosine-atriopeptin-21. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW030P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of ceUular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings, incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW030P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW030P

Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (anaritide) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching anaritide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oUgopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabUize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising anaritide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and anaritide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Anaritide preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW030P

the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting anaritide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering anaritide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, anaritide is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, anaritide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and anaritide is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, anaritide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, anaritide is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW030P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching anaritide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, anaritide and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW030P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize anaritide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of anaritide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises anaritide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW030P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be CW030P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH confroUed peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized, in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW030P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW030P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

u CW030P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, anaritide is covalently attached to the polypeptide via a peptide bond.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-fϊrst order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW030P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-anaritide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product

13 CW030P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

AIcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW030P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW030P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and anaritide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein anaritide is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW030P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein anaritide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing anaritide from said composition in a pH-dependent manner.

19. A method for protecting anaritide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of anaritide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching anaritide to said polypeptide.

21. A method for delivering anaritide to a patient comprising administering to said patient a composition comprising: a polypeptide; and anaritide covalently attached to said polypeptide.

22. The method of claim 21 wherein anaritide is released from said composition by an enzyme-catalyzed release.

17 CW030P

23. The method of claim 21 wherein anaritide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW030P

Abstract

A composition comprising a polypeptide and anaritide covalentiy attached to the polypeptide. Also provided is a method for delivery of anaritide to a patient comprising administering to the patient a composition comprising a polypeptide and anaritide covalently attached to the polypeptide. Also provided is a method for protecting anaritide from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of anaritide from a composition comprising covalently attaching it to the polypeptide.

19 CW031P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ANASTROZOLE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to anastrozole, as well as methods for protecting and administering anastrozole. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Anastrozole is a known pha naceutical agent that is used in the treatment of breast cancer. Its chemical name is alpha,alpha,alpha\alpha'-tetrarnethyl-5-(lH- 1,2,4- triazol-1 -ylmethyl)- 1,3-benzenediacetonitrile. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW031P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW031P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW031P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (anastrozole) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching anastrozole to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pha naceutical composition comprising anastrozole microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and anastrozole covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Anastrozole preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW031P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting anastrozole from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering anastrozole to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, anastrozole is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, anastrozole is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and anastrozole is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, anastrozole is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, anastrozole is released from the composition in a CW031P

sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching anastrozole to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, anastrozole and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW031P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent dehvery. First, the invention can stabilize anastrozole and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of anastrozole. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Anastrozole is the subject of EP 296749 B (1994), priority GB 14013 (1987), herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises anastrozole covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW031P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW031P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide wiU be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW031P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be confroUed.

As a practical example, the following table Usts the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW031P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW031P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW031P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-anastrozole conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW031P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW031P

Preparation of γ- Alky I Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW031P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and anastrozole covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein anastrozole is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW031P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein anastrozole is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing anastrozole from said composition in a pH-dependent manner.

19. A method for protecting anastrozole from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of anastrozole from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching anastrozole to said polypeptide.

21. A method for delivering anastrozole to a patient comprising administering to said patient a composition comprising: a polypeptide; and anastrozole covalently attached to said polypeptide.

22. The method of claim 21 wherein anastrozole is released from said composition by an enzyme-catalyzed release.

17 CW031P

23. The method of claim 21 wherein anastrozole is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW031P

Abstract

A composition comprising a polypeptide and anastrozole covalently attached to the polypeptide. Also provided is a method for delivery of anastrozole to a patient comprising administering to the patient a composition comprising a polypeptide and anastrozole covalently attached to the polypeptide. Also provided is a method for protecting anastrozole from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of anastrozole from a composition comprising covalently attaching it to the polypeptide.

19 CW032P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ANTISENSE OLIGONUCLEOTIDES AND METHODS OF MAKING AND

USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to antisense oligonucleotides, as well as methods for protecting and administering antisense oligonucleotides. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Antisense oligonucleotides are a class of compounds made of RNA that is complementary to the mRNA that produces a protein of interest. Their usefulness is primarily for gene therapy. Individual uses include those for the treatment of inflammatory bowel diseases (). They are made by chemical RNA synthesis or, alternatively, by using a gene constract containing the antisense orientation of the gene of interest and isolating the RNA of interest.

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW032P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stabuity of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabUity of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW032P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW032P

Particle size not only becomes a problem with injectable drags, as in the HAR apphcation, but absoφtion through the brush-border membrane of the intestines is Umited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (antisense oligonucleotides) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching antisense ohgonucleotides to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising antisense oligonucleotides microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and antisense oligonucleotides covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Antisense oligonucleotides preferably is covalently attached to a side chain, the

N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached CW032P

to the C-teoninus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting antisense oligonucleotides from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering antisense oligonucleotides to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefened embodiment, antisense oligonucleotides is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, antisense oligonucleotides is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and antisense oligonucleotides is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, antisense oligonucleotides is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, antisense oligonucleotides is released from the composition in a sustained release. In yet another prefened embodiment, the CW032P

composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching antisense oligonucleotides to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, antisense oligonucleotides and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is CW032P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize antisense oligonucleotides and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of antisense oligonucleotides. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises antisense oligonucleotides covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oUgopeptide, (u) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the CW032P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oh drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW032P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW032P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controUed.

As a practical example, the foUowing table Usts the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW032P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW032P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, antisense oligonucleotides are covalently attached to the polypeptide via the ribose hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW032P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-antisense oligonucleotides conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW032P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW032P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW032P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW032P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and antisense oligonucleotides covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein antisense oligonucleotides is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW032P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein antisense oligonucleotides is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing antisense oligonucleotides from said composition in a pH-dependent manner.

19. A method for protecting antisense oligonucleotides from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of antisense oligonucleotides from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching antisense oligonucleotides to said polypeptide.

21. A method for delivering antisense oligonucleotides to a patient comprising administering to said patient a composition comprising: a polypeptide; and antisense oligonucleotides covalently attached to said polypeptide.

22. The method of claim 21 wherein antisense oligonucleotides is released from said composition by an enzyme-catalyzed release. CW032P

23. The method of claim 21 wherein antisense oligonucleotides is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and antisense oligonucleotides covalently attached to the polypeptide. Also provided is a method for delivery of antisense oligonucleotides to a patient comprising administering to the patient a composition comprising a polypeptide and antisense oligonucleotides covalently attached to the polypeptide. Also provided is a method for protecting antisense oligonucleotides from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of antisense oligonucleotides from a composition comprising covalently attaching it to the polypeptide.

19 CW033P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ARIPIPRAZOLE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to aripiprazole, as well as methods for protecting and administering aripiprazole. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Aripiprazole is a known pharmaceutical agent that is used in reducing both the positive and negative symptoms of acutely psychotic patients. Its chemical name is 7-[4- [4-(2,3-dichlorophenyl)-l-piperazinyl]butoxy]-3,4-dihydro-2(lH)-quinolinone. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW033P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, wiU assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW033P

reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent avahable for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW033P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (aripiprazole) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching aripiprazole to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabdize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising aripiprazole microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and aripiprazole covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Aripiprazole preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW033P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting aripiprazole from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering aripiprazole to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, aripiprazole is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, aripiprazole is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and aripiprazole is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, aripiprazole is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, aripiprazole is released from the CW033P

composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching aripiprazole to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, aripiprazole and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW033P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize aripiprazole and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of aripiprazole. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Aripiprazole is the subject of EP 367141 B (1996)(priority Japan 276953 (1988)), herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises aripiprazole covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (u) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW033P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stabiUty is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW033P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW033P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW033P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW033P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of Unking organic compounds to the N-terminus type of a peptide include, but are not Umited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalentiy attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW033P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-aripiprazole conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW033P

AciόVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with dusopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW033P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AlkyI Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW033P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and aripiprazole covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein aripiprazole is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW033P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein aripiprazole is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing aripiprazole from said composition in a pH-dependent manner.

19. A method for protecting aripiprazole from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of aripiprazole from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching aripiprazole to said polypeptide.

21. A method for delivering aripiprazole to a patient comprising administering to said patient a composition comprising: a polypeptide; and aripiprazole covalently attached to said polypeptide.

22. The method of claim 21 wherein aripiprazole is released from said composition by an enzyme-catalyzed release.

17 CW033P

Abstract

A composition comprising a polypeptide and aripiprazole covalently attached to the polypeptide. Also provided is a method for delivery of aripiprazole to a patient comprising administering to the patient a composition comprising a polypeptide and aripiprazole covalentiy attached to the polypeptide. Also provided is a method for protecting aripiprazole from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controUing release of aripiprazole from a composition comprising covalently attaching it to the polypeptide.

19 CW034P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ASTEMIZOLE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to astemizole, as weU as methods for protecting and administering astemizole. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Astemizole is a known pharmaceutical agent that is used in the treatment of seasonal allergic rhinitis and chronic idiopathic urticara. Its chemical name is l-[(4- fluorophenyl)methyl] -N- [ 1 - [2-(4-methoxyphenyl)ethyl] -4-piperidinyl]- 1 H-benzimidazol- 2-amine. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical CW034P

compound may contain one or more of the following: another active phaonaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW034P

shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW034P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (astemizole) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching astemizole to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain appUcations, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising astemizole microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and astemizole covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occuning amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW034P

Astemizole preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting astemizole from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering astemizole to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, astemizole is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, astemizole is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and astemizole is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, astemizole is CW034P

released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, astemizole is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching astemizole to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, astemizole and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW034P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize astemizole and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of astemizole. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises astemizole covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the CW034P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW034P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW034P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW034P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicUlin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW034P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of Unking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW034P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-astemizole conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW034P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW034P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW034P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and astemizole covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein astemizole is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW034P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein astemizole is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing astemizole from said composition in a pH-dependent manner.

19. A method for protecting astemizole from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of astemizole from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching astemizole to said polypeptide.

21. A method for delivering astemizole to a patient comprising administering to said patient a composition comprising: a polypeptide; and astemizole covalently attached to said polypeptide.

22. The method of claim 21 wherein astemizole is released from said composition by an enzyme-catalyzed release.

17 CW034P

23. The method of claim 21 wherein astemizole is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW034P

Abstract

A composition comprising a polypeptide and astemizole covalently attached to the polypeptide. Also provided is a method for delivery of astemizole to a patient comprising administering to the patient a composition comprising a polypeptide and astemizole covalently attached to the polypeptide. Also provided is a method for protecting astemizole from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of astemizole from a composition comprising covalently attaching it to the polypeptide.

19 CW035P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ATENOLOL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to atenolol, as well as methods for protecting and administering atenolol. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of talcing a known effective phaonaceutical agent that is both well smdied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Atenolol is a known phaonaceutical agent that is used in the treatment of hypertension or chronic stable angina pectoris in patients with chronic obstructive pulmonary disease (COPD) or type 1 diabetes mellitus. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

CHgCONH;

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered CW035P

product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabiUty are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations. CW035P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag deUvery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW035P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (atenolol) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching atenolol to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising atenolol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and atenolol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW035P

heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Atenolol preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting atenolol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering atenolol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, atenolol is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, atenolol is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. CW035P

In another prefened embodiment, the composition further comprises a microencapsulating agent and atenolol is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, atenolol is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, atenolol is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching atenolol to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, atenolol and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW035P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize atenolol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of atenolol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises atenolol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oUgopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW035P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline soUd than an oil drop and so the best model for determining forces contributing to protein stabiUty is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stabiUty of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW035P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide wiU be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW035P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW035P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicUlin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW035P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, atenolol is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW035P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system wiU involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-atenolol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW035P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW035P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alk l Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW035P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and atenolol covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturaUy occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein atenolol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The coh position of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW035P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein atenolol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing atenolol from said composition in a pH-dependent manner.

19. A method for protecting atenolol from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of atenolol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching atenolol to said polypeptide.

21. A method for delivering atenolol to a patient comprising administering to said patient a composition comprising: a polypeptide; and atenolol covalently attached to said polypeptide.

22. The method of claim 21 wherein atenolol is released from said composition by an enzyme-catalyzed release.

17 CW035P

23. The method of claim 21 wherein atenolol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW035P

Abstract

A composition comprising a polypeptide and atenolol covalentiy attached to the polypeptide. Also provided is a method for delivery of atenolol to a patient comprising administering to the patient a composition comprising a polypeptide and atenolol covalently attached to the polypeptide. Also provided is a method for protecting atenolol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of atenolol from a composition comprising covalently attaching it to the polypeptide.

19 CW036P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ATORVASTATIN AND METHODS OF MAKING AND USING SAME

FTELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to atorvastatin, as well as methods for protecting and administering atorvastatin. This novel compound, refened to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well smdied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pha naceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Atorvastatin is a known pharmaceutical agent that is used in the treatment of high cholesterol. Its chemical name is (betaR,deltaR)-2-(4-fluorophenyl)-beta,delta- dihydroxy-5-( 1 -methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]- 1 H-pyoole- 1 - heptanoic acid. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW036P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibUity and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach πulieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW036P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW036P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (atorvastatin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching atorvastatin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising atorvastatin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and atorvastatin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Atorvastatin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW036P

carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting atorvastatin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering atorvastatin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, atorvastatin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, atorvastatin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and atorvastatin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, atorvastatin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, atorvastatin is released from the CW036P

composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching atorvastatin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, atorvastatin and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW036P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize atorvastatin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of atorvastatin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Atorvastatin is the subject of U.S. Patent Numbers 4,681,893, 5,273,995,

5,686,104, and 5,969,156, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises atorvastatin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring. amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW036P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW036P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW036P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW036P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oUgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW036P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not Umited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, atorvastatin is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW036P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system wUl involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-atorvastatin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW036P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW036P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW036P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and atorvastatin covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein atorvastatin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW036P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceuticaUy acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein atorvastatin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing atorvastatin from said composition in a pH-dependent manner.

19. A method for protecting atorvastatin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of atorvastatin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching atorvastatin to said polypeptide.

21. A method for delivering atorvastatin to a patient comprising administering to said patient a composition comprising: a polypeptide; and atorvastatin covalently attached to said polypeptide.

22. The method of claim 21 wherein atorvastatin is released from said composition by an enzyme-catalyzed release.

17 CW036P

23. The method of claim 21 wherein atorvastatin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW036P

Abstract

A composition comprising a polypeptide and atorvastatin covalently attached to the polypeptide. Also provided is a method for delivery of atorvastatin to a patient comprising administering to the patient a composition comprising a polypeptide and atorvastatin covalently attached to the polypeptide. Also provided is a method for protecting atorvastatin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of atorvastatin from a composition comprising covalently attaching it to the polypeptide.

19 CW037P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ATOVAQUONE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalentiy attached to atovaquone, as well as methods for protecting and administering atovaquone. This novel compound, refeoed to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pha naceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Atovaquone is a known pharmaceutical agent that is used in the prevention of Pneumocystis carinii pneumonia. Its chemical name is 2-[trans-4-(4- chlorophenyl)cyclohexyl]-3-hydroxy-l,4-naphthalenedione. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active_, pharmaceutical agent, an adjuvant, or an inhibitor. ^ CW037P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW037P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW037P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (atovaquone) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching atovaquone to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide wiU stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising atovaquone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and atovaquone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Atovaquone preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW037P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting atovaquone from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering atovaquone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, atovaquone is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, atovaquone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and atovaquone is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, atovaquone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, atovaquone is released from the composition in a CW037P

sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching atovaquone to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, atovaquone and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW037P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize atovaquone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of atovaquone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Atovaquone is the subject of U.S. Patent Numbers 4,981,874 and 5,053,432, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises atovaquone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW037P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW037P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of CW037P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW037P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW037P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, atovaquone is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW037P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of speciaUzed adjuvant resulting in localized deUvery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-atovaquone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW037P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW037P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW037P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and atovaquone covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein atovaquone is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW037P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein atovaquone is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing atovaquone from said composition in a pH-dependent manner.

19. A method for protecting atovaquone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of atovaquone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching atovaquone to said polypeptide.

21. A method for delivering atovaquone to a patient comprising administering to said patient a composition comprising: a polypeptide; and atovaquone covalently attached to said polypeptide.

22. The method of claim 21 wherein atovaquone is released from said composition by an enzyme-catalyzed release.

17 CW037P

23. The method of claim 21 wherein atovaquone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW037P

Abstract

A composition comprising a polypeptide and atovaquone covalently attached to the polypeptide. Also provided is a method for delivery of atovaquone to a patient comprising administering to the patient a composition comprising a polypeptide and atovaquone covalentiy attached to the polypeptide. Also provided is a method for protecting atovaquone from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of atovaquone from a composition comprising covalently attaching it to the polypeptide.

19 CW038P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AVASIMTBE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pha naceutical compound that comprises a polypeptide that is preferably covalently attached to avasimibe, as well as methods for protecting and administering avasimibe. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Avasimibe is a known pharmaceutical agent that is used in the treatment of hyperlipidemia. Its chemical name is N-[[2,6-bis(l-methylethyl)phenoxy]sulfonyl]- 2,4,6-tris(l-methylethyl)benzeneacetamide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW038P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bUayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW038P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW038P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (avasimibe) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching avasimibe to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising avasimibe microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and avasimibe covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Avasimibe preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW038P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting avasimibe from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering avasimibe to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, avasimibe is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, avasimibe is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and avasimibe is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, avasimibe is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, avasimibe is released from the composition in a sustained release. CW038P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching avasimibe to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, avasimibe and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW038P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize avasimibe and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of avasimibe. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Avasimibe is the subject of WO 94/26702 1994 (priority US 62515 (1993)), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises avasimibe covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW038P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are * "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW038P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW038P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug deUvery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controUed.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW038P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide Unked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW038P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not Umited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, avasimibe is covalently attached to the polypeptide via the sulfate group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW038P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-avasimibe conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW038P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW038P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW038P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and avasimibe covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oUgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein avasimibe is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW038P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein avasimibe is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing avasimibe from said composition in a pH-dependent manner.

19. A method for protecting avasimibe from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of avasimibe from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching avasimibe to said polypeptide.

21. A method for delivering avasimibe to a patient comprising administering to said patient a composition comprising: a polypeptide; and avasimibe covalently attached to said polypeptide.

22. The method of claim 21 wherein avasimibe is released from said composition by an enzyme-catalyzed release.

17 CW038P

23. The method of claim 21 wherein avasimibe is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW038P

Abstract

A composition comprising a polypeptide and avasimibe covalently attached to the polypeptide. Also provided is a method for deUvery of avasimibe to a patient comprising administering to the patient a composition comprising a polypeptide and avasimibe covalently attached to the polypeptide. Also provided is a method for protecting avasimibe from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of avasimibe from a composition comprising covalently attaching it to the polypeptide.

19 CW039P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AZATHIOPRENE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to azathioprene, as well as methods for protecting and administering azathioprene. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Azathioprene is a known pharmaceutical agent that is used in the treatment of transplant organ rejection. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

CW039P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted deUvery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach miUeu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW039P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stabUity and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent avaUable for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW039P

linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (azathioprene) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching azathioprene to the N-terminus, the C-teoninus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising azathioprene microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and azathioprene covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, CW039P

(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Azathioprene preferably is covalentiy attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teiminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting azathioprene from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering azathioprene to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the CW039P

polypeptide. In a prefened embodiment, azathioprene is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, azathioprene is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and azathioprene is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, azathioprene is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, azathioprene is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching azathioprene to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, azathioprene and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side CW039P

chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize azathioprene and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of azathioprene. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises azathioprene covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the poljφeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture. CW039P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW039P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide wUl be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but wiU ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW039P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW039P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oUgopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW039P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycyste ne, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or tinker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, azathioprene is covalently attached to the polypeptide via the nitrate group, or alternatively though a linker.

The polypeptide canier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW039P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-azathioprene conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW039P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW039P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW039P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW039P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and azathioprene covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein azathioprene is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW039P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein azathioprene is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing azathioprene from said composition in a pH-dependent manner.

19. A method for protecting azathioprene from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of azathioprene from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching azathioprene to said polypeptide.

21. A method for delivering azathioprene to a patient comprising administering to said patient a composition comprising: a polypeptide; and azathioprene covalently attached to said polypeptide.

22. The method of claim 21 wherein azathioprene is released from said composition by an enzyme-catalyzed release.

18 CW039P

23. The method of claim 21 wherein azathioprene is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and azathioprene covalently attached to the polypeptide. Also provided is a method for delivery of azathioprene to a patient comprising administering to the patient a composition comprising a polypeptide and azathioprene covalently attached to the polypeptide. Also provided is a method for protecting azathioprene from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of azathioprene from a composition comprising covalently attaching it to the polypeptide.

19 CW040P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AZELASTTNE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to azelastine, as well as methods for protecting and administering azelastine. This novel compound, refeoed to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Azelastine is a known pharmaceutical agent that is used in the treatment of itching of the eye associated with allergic conjunctivitis. Its chemical name is 4-[(4- chlorophenyl)methyl] -2-(hexahydro- 1 -methyl- 1 H-azepin-4-yl)- 1 (2H)-phthalazinone. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical CW040P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologicaUy active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabUity are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW040P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug deUvery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW040P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (azelastine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching azelastine to the N- teiminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, deUvery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising azelastine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and azelastine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW040P

Azelastine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting azelastine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering azelastine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, azelastine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, azelastine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and azelastine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, azelastine is released CW040P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, azelastine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of: (a) attaching azelastine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, azelastine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW040P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize azelastine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of azelastine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted deUvery of active agents to specifics sites of action.

Azelastine is the subject of U.S. Patent Number 5,164,194, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises azelastine covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iu) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW040P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW040P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but wUl ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW040P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug deUvery. GeneraUy, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an^{^}oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW040P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

11 CW040P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag deUvery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is teiminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW040P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system wiU involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-azelastine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW040P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW040P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water. ^*

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW040P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW040P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and azelastine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oUgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein azelastine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW040P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein azelastine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing azelastine from said composition in a pH-dependent manner.

19. A method for protecting azelastine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of azelastine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching azelastine to said polypeptide.

21. A method for delivering azelastine to a patient comprising administering to said patient a composition comprising: a polypeptide; and azelastine covalently attached to said polypeptide.

22. The method of claim 21 wherein azelastine is released from said composition by an enzyme-catalyzed release.

18 CW040P

23. The method of claim 21 wherein azelastine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and azelastine covalently attached to the polypeptide. Also provided is a method for delivery of azelastine to a patient comprising administering to the patient a composition comprising a polypeptide and azelastine covalently attached to the polypeptide. Also provided is a method for protecting azelastine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of azelastine from a composition comprising covalently attaching it to the polypeptide.

19 CW041P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AZITHROMYCIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to azithromycin, as well as methods for protecting and administering azithromycin. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Azithromycin is a known pharmaceutical agent that is used in the treatment of bacterial infections. Its chemical name is (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13- [(2,6-dideoxy-3-C-methyl-3-O-methyl-alpha-l-ribohexopyranosyl)oxy]-2-ethyl-3,4,10- trihy(froxy-3,5,6,8,10,12,heptamethyl-l l-[[3,4,6-trideoxy-3-(dimethylamino)beta-D- xylo-hexopyranosyl]oxy]-l-oxa-6-azacyclopentadecan-15-one. Its stracture is:

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability CW041P

of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf Ufe or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compUance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and fransport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified CW041P

amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that CW041P

incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (azithromycin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching azithromycin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oUgopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain appUcations, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising azithromycin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and azithromycin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a CW041P

heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Azithromycin preferably is covalentiy attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting azithromycin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering azithromycin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, azithromycin is released from the composition CW041P

by an enzyme-catalyzed release. In another prefeoed embodiment, azithromycin is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and azithromycin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, azithromycin is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, azithromycin is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching azithromycin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, azithromycin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a CW041P

carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize azithromycin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of azithromycin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Azithromycin is the subject of GB 2094293 B (1985), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises azithromycin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The CW041P

folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the sohd reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and CW041P

at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain CW041P

length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

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TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

11 CW041P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW041P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, azithromycin is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW041P

mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-azithromycin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

14 CW041P

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW041P

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW041P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and azithromycin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oUgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein azithromycin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW041P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein azithromycin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing azithromycin from said composition in a pH-dependent manner.

19. A method for protecting azithromycin from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of azithromycin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching azithromycin to said polypeptide.

21. A method for delivering azithromycin to a patient comprising administering to said patient a composition comprising: a polypeptide; and azithromycin covalently attached to said polypeptide.

22. The method of claim 21 wherein azithromycin is released from said composition by an enzyme-catalyzed release.

18 CW041P

23. The method of claim 21 wherein azithromycin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and azithromycin covalentiy attached to the polypeptide. Also provided is a method for delivery of azithromycin to a patient comprising administering to the patient a composition comprising a polypeptide and azithromycin covalently attached to the polypeptide. Also provided is a method for protecting azithromycin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of azithromycin from a composition comprising covalently attaching it to the polypeptide.

19 CW042P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BACLOFEN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to baclofen, as well as methods for protecting and administering baclofen. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Baclofen is a known pharmaceutical agent that is used in the treatment of spasticity. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW042P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW042P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typicaUy require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: Unking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW042P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (baclofen) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching baclofen to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising baclofen microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and baclofen covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Baclofen preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW042P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-teiminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- teπninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting baclofen from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering baclofen to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, baclofen is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, baclofen is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and baclofen is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, baclofen is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, baclofen is released from the composition in a sustained release. CW042P

In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching baclofen to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, baclofen and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW042P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize baclofen and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of baclofen. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises baclofen covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (Ui) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW042P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stabiUty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW042P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophihcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW042P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teoninus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW042P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, baclofen is covalently attached to the polypeptide via the carboxylic acid group or the amine group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW042P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oUgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-baclofen conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW042P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoohdinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW042P

Preparation of γ- lkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodUmide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW042P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and baclofen covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein baclofen is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW042P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein baclofen is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said poljφeptide is capable of releasing baclofen from said composition in a pH-dependent manner.

19. A method for protecting baclofen from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controUing release of baclofen from a composition wherein said composition comprises a poljφeptide, said method comprising covalently attaching baclofen to said polypeptide.

21. A method for delivering baclofen to a patient comprising administering to said patient a composition comprising: a polypeptide; and baclofen covalently attached to said polypeptide.

22. The method of claim 21 wherein baclofen is released from said composition by an enzyme-catalyzed release.

17 CW042P

23. The method of claim 21 wherein baclofen is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW042P

Abstract

A composition comprising a polypeptide and baclofen covalentiy attached to the polypeptide. Also provided is a method for delivery of baclofen to a patient comprising administering to the patient a composition comprising a polypeptide and baclofen covalently attached to the polypeptide. Also provided is a method for protecting baclofen from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of baclofen from a composition comprising covalently attaching it to the poljφeptide.

19 CW043P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BEFLOXATONE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to befloxatone, as well as methods for protecting and administering befloxatone. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Befloxatone is a known phaimaceutical agent that is used in smoking cessation treatment. Its chemical name is (R)-5-(methoxymethyl)-3-[4-[(R)-4,4,4-trifluoro-3- hydroxybutoxy)phenyl]-2-oxazolidinone. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor. CW043P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabiUty of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW043P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typicaUy require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug deUvery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW043P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (befloxatone) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching befloxatone to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabhize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising befloxatone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and befloxatone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Befloxatone preferably is covalently attached to a side chain, the N-teoninus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW043P

carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting befloxatone from degradation comprising covalentiy attaching it to a poljφeptide.

The invention also provides a method for delivering befloxatone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefeoed embodiment, befloxatone is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, befloxatone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and befloxatone is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, befloxatone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, befloxatone is released from the CW043P

composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching befloxatone to a side chain of an amino acid to form an active agent amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, befloxatone and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW043P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize befloxatone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of befloxatone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Befloxatone is the subject of EP 424244 B (1995), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises befloxatone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW043P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW043P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW043P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW043P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW043P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, befloxatone is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW043P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized deUvery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-befloxatone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW043P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine. dimethylaminopyridine, triethylamine or tributylamine.

14 CW043P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW043P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and befloxatone covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide. -

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said poljφeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein befloxatone is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW043P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein befloxatone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing befloxatone from said composition in a pH-dependent manner.

19. A method for protecting befloxatone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of befloxatone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching befloxatone to said polypeptide.

21. A method for delivering befloxatone to a patient comprising administering to said patient a composition comprising: a polypeptide; and befloxatone covalently attached to said polypeptide.

22. The method of claim 21 wherein befloxatone is released from said composition by an enzyme-catalyzed release.

17 CW043P

23. The method of claim 21 wherein befloxatone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW043P

Abstract

A composition comprising a polypeptide and befloxatone covalently attached to the polypeptide. Also provided is a method for delivery of befloxatone to a patient comprising administering to the patient a composition comprising a polypeptide and befloxatone covalently attached to the polypeptide. Also provided is a method for protecting befloxatone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of befloxatone from a composition comprising covalently attaching it to the polypeptide.

19 CW044P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BENAZEPRIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to benazepril, as well as methods for protecting and administering benazepril. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Benazepril has the chemical name [S-(R*,R*)]-3-[[l-(ethoxycarbonyl)-3- phenylpropyl]amino]-2,3,4,5-tetrahydro-2-oxo-lH-l-benzazepine-l-acetic acid. It is available commercially or can be made by those of skill in the art. Its structure is as follows:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered CW044P

product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabiUty are taken under consideration. For instance, one would expect patient compUance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabUity of cellular membranes and fransport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent deUvery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations. CW044P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and Unking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW044P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (benazepril) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching benazepril to the N- teπninus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the poljφeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising benazepril microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and benazepril covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW044P

heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Benazepril preferably is covalently attached to a side chain, the N-teoninus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting benazepril from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering benazepril to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, benazepril is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, benazepril is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed CW044P

release. In another prefened embodiment, the composition further comprises a microencapsulating agent and benazepril is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, benazepril is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, benazepril is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching benazepril to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, benazepril and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW044P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize benazepril and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of benazepril. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also aUows targeted delivery of active agents to specifics sites of action.

Benazepril is the subject of U.S. Patent Number 4,410,520, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises benazepril covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oUgopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture. CW044P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more Uke a crystalline solid than an oU drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW044P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW044P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW044P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oUgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oUgopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW044P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig.4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-teoninus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, benazepril is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW044P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-benazepril conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW044P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW044P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AlkyI Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW044P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW044P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and benazepril covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein benazepril is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW044P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein benazepril is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing benazepril from said composition in a pH-dependent manner.

19. A method for protecting benazepril from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of benazepril from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching benazepril to said polypeptide.

21. A method for delivering benazepril to a patient comprising administering to said patient a composition comprising: a poljφeptide; and benazepril covalently attached to said polypeptide.

22. The method of claim 21 wherein benazepril is released from said composition by an enzyme-catalyzed release.

18 CW044P

23. The method of claim 21 wherein benazepril is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and benazepril covalently attached to the polypeptide. Also provided is a method for delivery of benazepril to a patient comprising administering to the patient a composition comprising a polypeptide and benazepril covalently attached to the polypeptide. Also provided is a method for protecting benazepril from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of benazepril from a composition comprising covalently attaching it to the polypeptide.

19 CW045P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BENZATROPINE MESYLATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to benzatropine mesylate, as well as methods for protecting and administering benzatropine mesylate. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Benzatropine mesylate is a known phaimaceutical agent that is used in the treatment of Parkinsoniajn syndrome. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW045P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and fransport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of ceUular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW045P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typicaUy require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW045P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (benzatropine mesylate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching benzatropine mesylate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide wiU stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising benzatropine mesylate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and benzafropine mesylate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Benzatropine mesylate preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active CW045P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting benzatropine mesylate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering benzafropine mesylate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, benzatropine mesylate is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, benzatropine mesylate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and benzafropine mesylate is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, benzatropine mesylate is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, benzatropine CW045P

mesylate is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching benzatropine mesylate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, benzatropine mesylate and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW045P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize benzafropine mesylate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of benzatropine mesylate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises benzatropine mesylate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (U) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the CW045P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that hpophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioe^'versible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW045P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but wiU ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW045P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag deUvery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW045P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-teoninus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW045P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not Umited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the poljφeptide using known techniques. Examples of Unking organic compounds to the N-terminus type of a peptide include, but are not Umited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, benzatropine mesylate is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW045P

The addition of stabilizers to the composition has the potential of stabiUzing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized deUvery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oUgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to faciUtate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-benzatropine mesylate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW045P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW045P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW045P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and benzafropine mesylate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein benzatropine mesylate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW045P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein benzafropine mesylate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing benzatropine mesylate from said composition in a pH-dependent manner.

19. A method for protecting benzatropine mesylate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of benzatropine mesylate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching benzatropine mesylate to said polypeptide.

21. A method for delivering benzatropine mesylate to a patient comprising administering to said patient a composition comprising: a polypeptide; and benzatropine mesylate covalently attached to said polypeptide.

22. The method of claim 21 wherein benzatropine mesylate is released from said composition by an enzyme-catalyzed release.

17 CW045P

23. The method of claim 21 wherein benzafropine mesylate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW046P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BETAMETHASONE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to betamethasone, as well as methods for protecting and administering betamethasone. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Betamethasone is a known pharmaceutical agent that is used principally as an anti-inflammatory or immunosuppressant agent. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW046P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and fransport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW046P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW046P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (betamethasone) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching betamethasone to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising betamethasone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and betamethasone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW046P

Betamethasone preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting betamethasone from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering betamethasone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, betamethasone is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, betamethasone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and betamethasone is released from the composition by dissolution of the microencapsulating agent. In another prefeoed CW046P

embodiment, betamethasone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, betamethasone is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching betamethasone to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, betamethasone and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW046P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize betamethasone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of betamethasone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted deUvery of active agents to specifics sites of action.

The composition of the invention comprises betamethasone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (Ui) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurong amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW046P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW046P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the canier polypeptide. Another, significant advantage of the invention is that the kinetics of CW046P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW046P

delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-teoninus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW046P

any suitable initiator as shown in Fig.4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, betamethasone is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW046P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier poljφeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-betamethasone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW046P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylaπύne or tributylamine.

14 CW046P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW046P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and betamethasone covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oUgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein betamethasone is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW046P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein betamethasone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing betamethasone from said composition in a pH-dependent manner.

19. A method for protecting betamethasone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of betamethasone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching betamethasone to said polypeptide.

21. A method for delivering betamethasone to a patient comprising administering to said patient a composition comprising: a polypeptide; and betamethasone covalently attached to said polypeptide.

22. The method of claim 21 wherein betamethasone is released from said composition by an enzyme-catalyzed release.

17 CW046P

23. The method of claim 21 wherein betamethasone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW046P

Abstract

A composition comprising a polypeptide and betamethasone covalently attached to the polypeptide. Also provided is a method for delivery of betamethasone to a patient comprising administering to the patient a composition comprising a polypeptide and betamethasone covalently attached to the polypeptide. Also provided is a method for protecting betamethasone from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of betamethasone from a composition comprising covalently attaching it to the polypeptide.

19 CW047P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BICALUTAMIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to bicalutamide, as well as methods for protecting and administering bicalutamide. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Bicalutamide is a known pharmaceutical agent that is used in the treatment of locally advanced, non-metastatic prostate cancer, in combination with LHRH. Its chemical name is (+,-)-N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4- fluorophenyl)sulfonyl]-2-hydroxy-2-methylpropanamide. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW047P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW047P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW047P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (bicalutamide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching bicalutamide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising bicalutamide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and bicalutamide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Bicalutamide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW047P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting bicalutamide from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering bicalutamide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, bicalutamide is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, bicalutamide is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and bicalutamide is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, bicalutamide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, bicalutamide is released from the CW047P

composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching bicalutamide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, bicalutamide and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW047P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize bicalutamide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of bicalutamide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Bicalutamide is the subject of U.S. Patent Numbers 4,472,382, 4,636,505, and

5,389,613, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises bicalutamide covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW047P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW047P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophihcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier poljφeptide. Another, significant advantage of the invention is that the kinetics of CW047P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tjrrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW047P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW047P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, bicalutamide is covalently attached to the polypeptide via the hydroxyl group. LHRH may also be attached to the same polypeptide to provide the two drags in combination.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW047P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-bicalutamide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW047P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW047P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alky I Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW047P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and bicalutamide covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein bicalutamide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW047P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein bicalutamide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing bicalutamide from said composition in a pH-dependent manner.

19. A method for protecting bicalutamide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of bicalutamide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching bicalutamide to said polypeptide.

21. A method for delivering bicalutamide to a patient comprising administering to said patient a composition comprising: a polypeptide; and bicalutamide covalently attached to said polypeptide.

22. The method of claim 21 wherein bicalutamide is released from said composition by an enzyme-catalyzed release.

17 CW047P

23. The method of claim 21 wherein bicalutamide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW047P

Abstract

A composition comprising a polypeptide and bicalutamide covalently attached to the polypeptide. Also provided is a method for delivery of bicalutamide to a patient comprising administering to the patient a composition comprising a polypeptide and bicalutamide covalently attached to the polypeptide. Also provided is a method for protecting bicalutamide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of bicalutamide from a composition comprising covalently attaching it to the polypeptide.

19 CW048P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BISOPROLOL

AND HYDROCHLOROTHIAZIDE AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to bisoprolol and hydrochlorothiazide, as weU as methods for protecting and administering bisoprolol and hydrochlorothiazide. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Bisoprolol and hydrochlorothiazide is a known phaimaceutical agent that is used in the treatment of . Its chemical name is . Its structure is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW048P

invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across Upid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW048P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drug conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug deUvery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films Otnown as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW048P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (bisoprolol and hydrochlorothiazide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching bisoprolol and hydrochlorothiazide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabUize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzjmπatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising bisoprolol and hydrochlorothiazide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and bisoprolol and hydrochlorothiazide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Bisoprolol and hydrochlorothiazide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus CW048P

of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting bisoprolol and hydrochlorothiazide from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering bisoprolol and hydrochlorothiazide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the poljφeptide. In a prefened embodiment, bisoprolol and hydrochlorothiazide is released from the composition by an enzyme- catalyzed release. In another prefened embodiment, bisoprolol and hydrochlorothiazide is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and bisoprolol and hydrochlorothiazide is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, bisoprolol and hydrochlorothiazide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, bisoprolol and hydrochlorothiazide is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently CW048P

attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching bisoprolol and hydrochlorothiazide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, bisoprolol and hydrochlorothiazide and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW048P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize bisoprolol and hydrochlorothiazide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of bisoprolol and hydrochlorothiazide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted deUvery of active agents to specifics sites of action.

Bisoprolol and hydrochlorothiazide is the subject of U.S. Patent Number yyyyy, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises bisoprolol and hydrochlorothiazide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding CW048P

are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW048P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW048P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW048P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

11 CW048P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-teoninus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of Unking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, bisoprolol and hydrochlorothiazide is covalently attached to the polypeptide via the zzzzzzz.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW048P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier poljφeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system wiU involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithehal transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-bisoprolol and hydrochlorothiazide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW048P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW048P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW048P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW048P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and bisoprolol and hydrochlorothiazide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein bisoprolol and hydrochlorothiazide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW048P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceuticaUy acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein bisoprolol and hydrochlorothiazide is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing bisoprolol and hydrochlorothiazide from said composition in a pH-dependent manner.

19. A method for protecting bisoprolol and hydrochlorothiazide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of bisoprolol and hydrochlorothiazide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching bisoprolol and hydrochlorothiazide to said polypeptide.

21. A method for delivering bisoprolol and hydrochlorothiazide to a patient comprising administering to said patient a composition comprising: a polypeptide; and bisoprolol and hydrochlorothiazide covalently attached to said polypeptide.

22. The method of claim 21 wherein bisoprolol and hydrochlorothiazide is released from said composition by an enzyme-catalyzed release.

18 CW048P

23. The method of claim 21 wherein bisoprolol and hydrochlorothiazide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

19 CW048P

Abstract

A composition comprising a polypeptide and bisoprolol and hydrochlorothiazide covalently attached to the polypeptide. Also provided is a method for delivery of bisoprolol and hydrochlorothiazide to a patient comprising administering to the patient a composition comprising a poljφeptide and bisoprolol and hydrochlorothiazide covalently attached to the polypeptide. Also provided is a method for protecting bisoprolol and hydrochlorothiazide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controUing release of bisoprolol and hydrochlorothiazide from a composition comprising covalently attaching it to the polypeptide.

20 CW049P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BOSENTAN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to bosentan, as well as methods for protecting and administering bosentan. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Bosentan is a known pharmaceutical agent that is used in the treatment of pulmonary hypertension. Its chemical name is 4-(l,l-dimethylethyl)-N-[6-(2- hydroxyethoxy)-5-(2-methoxjφhenoxy)[2,2'-bipjτinMdin]-4-yl]benzenesulfonamide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW049P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW049P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW049P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (bosentan) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching bosentan to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent', primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enz matic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising bosentan microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and bosentan covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW049P

Bosentan preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting bosentan from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering bosentan to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, bosentan is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, bosentan is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and bosentan is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, bosentan is released CW049P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, bosentan is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching bosentan to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, bosentan and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW049P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabihze bosentan and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of bosentan. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Bosentan is the subject of EP 526708 A (1993), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises bosentan covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (Ui) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW049P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stabiUty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW049P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydropbilicity of the poljφeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW049P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW049P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teoninus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW049P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not Umited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, pol ythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, bosentan is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW049P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oUgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-bosentan conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW049P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel peoneation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

AIcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW049P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW049P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and bosentan covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturaUy occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said poljφeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein bosentan is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW049P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein bosentan is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing bosentan from said composition in a pH-dependent manner.

19. A method for protecting bosentan from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of bosentan from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching bosentan to said polypeptide.

21. A method for delivering bosentan to a patient comprising administering to said patient a composition comprising: a polypeptide; and bosentan covalently attached to said polypeptide.

22. The method of claim 21 wherein bosentan is released from said composition by an enzyme-catalyzed release.

17 CW049P

23. The method of claim 21 wherein bosentan is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW049P

Abstract

A composition comprising a polypeptide and bosentan covalently attached to the polypeptide. Also provided is a method for deUvery of bosentan to a patient comprising administering to the patient a composition comprising a polypeptide and bosentan covalently attached to the polypeptide. Also provided is a method for protecting bosentan from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of bosentan from a composition comprising covalentiy attaching it to the poljφeptide.

19 CW050P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BROMOCRIPTTNE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to bromocriptine, as well as methods for protecting and administering bromocriptine. This novel compound, refened to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Bromocriptine is a known pha naceutical agent that is used in the treatment of dysfunctions associated with hypeφrolactinemia including amenoohea, with or without galactoohea; hypogonadism; and infertility. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW050P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabUity are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach miUeu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stabihty and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW050P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typicaUy require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW050P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (bromocriptine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching bromocriptine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or poljφeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising bromocriptine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and bromocriptine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW050P

Bromocriptine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting bromocriptine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering bromocriptine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, bromocriptine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, bromocriptine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and bromocriptine is released from the composition by dissolution of the microencapsulating agent. In another prefened CW050P

embodiment, bromocriptine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, bromocriptine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching bromocriptine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, bromocriptine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW050P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETATT.KD DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize bromocriptine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of bromocriptine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises bromocriptine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oUgopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW050P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW050P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophiUcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW050P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the foUowing table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

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delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

^' The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW050P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-teoninus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, bromocriptine is covalently attached to the polypeptide via the hydroxyl group. Alternatively, it is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

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Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epitheUal transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-bromocriptine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW050P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW050P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alky 1 Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW050P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW050P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and bromocriptine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturaUy occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein bromocriptine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW050P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein bromocriptine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing bromocriptine from said composition in a pH-dependent manner.

19. A method for protecting bromocriptine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of bromocriptine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching bromocriptine to said polypeptide.

21. A method for delivering bromocriptine to a patient comprising administering to said patient a composition comprising: a polypeptide; and bromocriptine covalently attached to said polypeptide.

22. The method of claim 21 wherein bromocriptine is released from said composition by an enzyme-catalyzed release.

18 CW050P

23. The method of claim 21 wherein bromocriptine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said poljφeptide.

Abstract

A composition comprising a polypeptide and bromocriptine covalently attached to the polypeptide. Also provided is a method for delivery of bromocriptine to a patient comprising administering to the patient a composition comprising a polypeptide and bromocriptine covalently attached to the polypeptide. Also provided is a method for protecting bromocriptine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for confrolUng release of bromocriptine from a composition comprising covalently attaching it to the polypeptide.

19 CW051P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BUPROPIONAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to bupropion, as well as methods for protecting and administering bupropion. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Bupropion is a known pharmaceutical agent that is used in smoking cessation therapy and in the treatment of depression. Its chemical name is l-(3-chlorophenyl)-2- [(1,1 -dimethylethyl)amino]- 1 -propanone. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW051P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabUity of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW051P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW051P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(bupropion) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching bupropion to the N- terminus, the C-teoninus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising bupropion microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and bupropion covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Bupropion preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW051P

the C-terminus of the poljφeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting bupropion from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering bupropion to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, bupropion is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, bupropion is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and bupropion is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, bupropion is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, bupropion is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW051P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching bupropion to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, bupropion and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Apphcation Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW051P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize bupropion and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of bupropion. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Bupropion is the subject of U.S. Patent Numbers 5,358,970, 5,427,798, 5,731,000, 5,763,493, and Re. 33,994, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises bupropion covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the CW051P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stabiUty is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW051P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but wUl ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW051P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW051P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-teoninus, the C-teoninus or the side chain of the oUgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oUgopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to polyO ydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW051P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, bupropion is covalently attached to the polypeptide via the amino group. Alternatively, it is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW051P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetricaUy to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized deUvery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the fransported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-bupropion conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW051P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW051P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW051P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW051P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and bupropion covalently attached to said polypeptide.

2. The composition of claim 1 wherein said poljφeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein bupropion is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW051P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein bupropion is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing bupropion from said composition in a pH-dependent manner.

19. A method for protecting bupropion from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of bupropion from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching bupropion to said polypeptide.

21. A method for delivering bupropion to a patient comprising administering to said patient a composition comprising: a polypeptide; and bupropion covalently attached to said polypeptide.

22. The method of claim 21 wherein bupropion is released from said composition by an enzyme-catalyzed release.

18 CW051P

23. The method of claim 21 wherein bupropion is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and bupropion covalently attached to the polypeptide. Also provided is a method for delivery of bupropion to a patient comprising administering to the patient a composition comprising a poljφeptide and bupropion covalentiy attached to the polypeptide. Also provided is a method for protecting bupropion from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of bupropion from a composition comprising covalently attaching it to the polypeptide.

19 CW052P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BUSPIRONE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to buspirone, as well as methods for protecting and administering buspirone. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Buspirone is a known pharmaceutical agent that is used in the treatment of atopic dermatitis. Its chemical name is 8-[4-[4-(2-pyrimidinyl)-l-piperazinyl]butyl]-8- azaspiro[4,5]decane-7,9-dione hydrochloride. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW052P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of ceUular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW052P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: Unking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW052P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brash-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (buspirone) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching buspirone to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising buspirone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and buspirone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Buspirone preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW052P

carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting buspirone from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering buspirone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefened embodiment, buspirone is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, buspirone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and buspirone is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, buspirone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, buspirone is released from the composition in a sustained release. CW052P

In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching buspirone to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, buspirone and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW052P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize buspirone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of buspirone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also aUows targeted delivery of active agents to specifics sites of action.

Buspirone is the subject of U.S. Patent Number 4,182,763 and 5,015,646, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises buspirone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (U) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW052P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stabUity of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW052P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW052P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW052P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. .The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW052P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW052P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-buspirone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW052P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW052P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW052P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and buspirone covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein buspirone is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW052P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein buspirone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing buspirone from said composition in a pH-dependent manner.

19. A method for protecting buspirone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of buspirone from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching buspirone to said polypeptide.

21. A method for delivering buspirone to a patient comprising administering to said patient a composition comprising: a polypeptide; and buspirone covalently attached to said polypeptide.

22. The method of claim 21 wherein buspirone is released from said composition by an enzyme-catalyzed release.

17 CW052P

23. The method of claim 21 wherein buspirone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW052P

Abstract

A composition comprising a polypeptide and buspirone covalently attached to the polypeptide. Also provided is a method for delivery of buspirone to a patient comprising administering to the patient a composition comprising a polypeptide and buspirone covalently attached to the polypeptide. Also provided is a method for protecting buspirone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of buspirone from a composition comprising covalently attaching it to the polypeptide.

19 CW053P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING BUTORPHANOL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to butoφhanol, as weU as methods for protecting and administering butoφhanol. This novel compound, refeoed to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Butoφhanol is a known phaonaceutical agent that is used in the treatment of pain. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; CW053P

and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations. CW053P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW053P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (butoφhanol) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching butoφhanol to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising butoφhanol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and butoφhanol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW053P

heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Butoφhanol preferably is covalentiy attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting butoφhanol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering butoφhanol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, butoφhanol is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, butoφhanol is released in a time-dependent manner based on the pharmacokinetics of the enzyme- CW053P

catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and butoφhanol is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, butoφhanol is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, butoφhanol is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching butoφhanol to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, butoφhanol and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the CW053P

glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize butoφhanol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of butoφhanol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises butoφhanol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture. CW053P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW053P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids wUl depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier poljφeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW053P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW053P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached ,to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW053P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be poljrmerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polyt TOsine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, butoφhanol is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW053P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-butoφhanol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW053P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW053P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW053P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW053P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and butoφhanol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said poljφeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein butoφhanol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW053P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein butoφhanol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing butoφhanol from said composition in a pH-dependent manner.

19. A method for protecting butoφhanol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of butoφhanol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching butoφhanol to said polypeptide.

21. A method for delivering butoφhanol to a patient comprising administering to said patient a composition comprising: a polypeptide; and butoφhanol covalently attached to said polypeptide.

22. The method of claim 21 wherein butoφhanol is released from said composition by an enzyme-catalyzed release.

18 CW053P

23. The method of claim 21 wherein butoφhanol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and butoφhanol covalently attached to the polypeptide. Also provided is a method for delivery of butoφhanol to a patient comprising administering to the patient a composition comprising a polypeptide and butoφhanol covalentiy attached to the polypeptide. Also provided is a method for protecting butoφhanol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of butoφhanol from a composition comprising covalently attaching it to the polypeptide.

19 CW054P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CABERGOLINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to cabergoline, as well as methods for protecting and administering cabergoline. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cabergoline is a known phaimaceutical agent that is used in the treatment of Parkinson's disease. Its chemical name is (8beta)-N-[3-(dimethylamino)propyl]-N- [(ethylamino)carbonyl]-6-(2-prop enyl)ergoline-8-carboxamide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stabihty of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW054P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeabiUty of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabiUty of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW054P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW054P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cabergoline) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cabergoline to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cabergoline microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cabergoline covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cabergoline preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW054P

carboxylic acid and is covalently attached to the N-teoninus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cabergoline from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cabergoline to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, cabergoline is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cabergoline is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cabergoline is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cabergoline is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cabergoline is released from the CW054P

composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cabergoline to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cabergoline and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW054P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cabergoline and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cabergoline. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cabergoline is the subject of U.S. Patent Number 4,526,892, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cabergoline covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturaUy occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW054P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stabiUty is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW054P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW054P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be confroUed.

As a practical example, the following table Usts the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW054P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampiciUin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchlorofoonate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly fooned free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW054P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cabergoline is covalently attached to the polypeptide via the amino group. Alternatively, it can be covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW054P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cabergoline conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW054P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Ai ne/C-ternύnus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW054P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alk l Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW054P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW054P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cabergoline covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturaUy occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cabergoline is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW054P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceuticaUy acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cabergoline is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cabergoline from said composition in a pH-dependent manner.

19. A method for protecting cabergoline from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of cabergoline from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cabergoline to said polypeptide.

21. A method for delivering cabergoline to a patient comprising administering to said patient a composition comprising: a polypeptide; and cabergoline covalently attached to said polypeptide.

22. The method of claim 21 wherein cabergoline is released from said composition by an enzyme-catalyzed release.

18 CW054P

23. The method of claim 21 wherein cabergoline is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and cabergoline covalentiy attached to the polypeptide. Also provided is a method for delivery of cabergoline to a patient comprising administering to the patient a composition comprising a polypeptide and cabergoline covalently attached to the polypeptide. Also provided is a method for protecting cabergoline from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cabergoline from a composition comprising covalently attaching it to the polypeptide.

19 CW055P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CAFFEINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to caffeine, as well as methods for protecting and administering caffeine. This novel compound, refeoed to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Caffeine is a known pharmaceutical agent that is used in the treatment of neonatal apnea. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; CW055P

and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibiUty and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations. CW055P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibUity. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: Unking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone _^active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW055P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexfran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labUe active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (caffeine) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalentiy attaching caffeine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is confroUed, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising caffeine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and caffeine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW055P

heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Caffeine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teiminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting caffeine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering caffeine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, caffeine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, caffeine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. CW055P

In another prefened embodiment, the composition further comprises a microencapsulating agent and caffeine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, caffeine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, caffeine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching caffeine to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, caffeine and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW055P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize caffeine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of caffeine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises caffeine covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW055P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oU drop and so the best model for determining forces contributing to protein stabiUty is the sohd reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophUic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW055P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophihcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW055P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW055P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW055P

any suitable initiator as shown in Fig.4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-teoninus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalentiy attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW055P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-caffeine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW055P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW055P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

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What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and caffeine covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said poljφeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein caffeine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein caffeine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing caffeine from said composition in a pH-dependent manner.

19. A method for protecting caffeine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of caffeine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching caffeine to said polypeptide.

21. A method for delivering caffeine to a patient comprising administering to said patient a composition comprising: a polypeptide; and caffeine covalently attached to said polypeptide.

22. The method of claim 21 wherein caffeine is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein caffeine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW055P

Abstract

A composition comprising a polypeptide and caffeine covalently attached to the polypeptide. Also provided is a method for delivery of caffeine to a patient comprising administering to the patient a composition comprising a polypeptide and caffeine covalentiy attached to the polypeptide. Also provided is a method for protecting caffeine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of caffeine from a composition comprising covalentiy attaching it to the polypeptide.

19 CW056P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CALCITRIOL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to calcitriol, as well as methods for protecting and administering calcitriol. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Calcitriol is a known phaonaceutical agent that is used in the treatment of hypocalcemia. Its stracture is:

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active phaonaceutical agent, an adjuvant, or an inhibitor. CW056P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf Ufe or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW056P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW056P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (calcitriol) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching calcitriol to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising calcitriol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and calcitriol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an ohgopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Calcitriol preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW056P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting calcitriol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering calcitriol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, calcitriol is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, calcitriol is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and calcitriol is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, calcitriol is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, calcitriol is released from the composition in a sustained release. CW056P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching calcitriol to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, calcitriol and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW056P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize calcitriol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of calcitriol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Calcitriol is the subject of U.S. Patent Numbers 4,308,264 and 6,051 ,567, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises calcitriol covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW056P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stabiUty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW056P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW056P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of Upophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW056P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oUgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teπriinus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW056P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, calcitriol is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW056P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized fransporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-calcitriol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW056P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW056P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW056P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and calcitriol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said poljφeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein calcitriol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein calcitriol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing calcitriol from said composition in a pH-dependent manner.

19. A method for protecting calcitriol from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of calcitriol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching calcitriol to said polypeptide.

21. A method for delivering calcitriol to a patient comprising administering to said patient a composition comprising: a polypeptide; and calcitriol covalently attached to said polypeptide.

22. The method of claim 21 wherein calcitriol is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein calcitriol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW056P

Abstract

A composition comprising a polypeptide and calcitriol covalently attached to the polypeptide. Also provided is a method for delivery of calcitriol to a patient comprising administering to the patient a composition comprising a polypeptide and calcitriol covalentiy attached to the polypeptide. Also provided is a method for protecting calcitriol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of calcitriol from a composition comprising covalentiy attaching it to the polypeptide.

19 CW057P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CANDESARTAN CILEXITIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to candesartan cilexitil, as well as methods for protecting and administering candesartan cilexitil. This novel compound, refeoed to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Candesartan cilexitil is a known phaonaceutical agent that is used in the treatment of heart failure. Its chemical name is 2-ethoxy-l-[[2'-(lH-tetrazol-5-yl)[l,l'-biphenyl]-4- yl]methyl]-l-H-benzimidazole-7-carboxylic acid l-[[(cyclohexyloxy)carbonyl]oxy]ethyl ester. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW057P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stabUity of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibUity and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW057P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW057P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (candesartan cilexitil) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching candesartan cilexitil to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising candesartan cilexitil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and candesartan cilexitil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW057P

Candesartan cilexitil preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting candesartan cilexitil from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering candesartan cilexitil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, candesartan cilexitil is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, candesartan cilexitil is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and candesartan cilexitil is released from the composition by dissolution of the microencapsulating agent. In another CW057P

prefened embodiment, candesartan cilexitil is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefened embodiment, candesartan cilexitil is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching candesartan cilexitil to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, candesartan cilexitil and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW057P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize candesartan cilexitil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of candesartan cilexitil. Furthermore, active agents can be combined to produce synergistic effects.

Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted deUvery of active agents to specifics sites of action.

Candesartan cilexitil is the subject of U.S. Patent Numbers 5,196,444, 5,534,534, 5,703,110 and 5,705,517, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises candesartan cilexitil covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure. CW057P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW057P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Upophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the Unear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW057P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW057P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW057P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, rrølythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

12 CW057P

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophUic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

13 CW057P

Preferably, the resultant peptide-candesartan cilexitil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated

14 CW057P

solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyπolidinopyridine, dimethylaminopyridine, ttiethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

15 CW057P

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW057P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and candesartan cilexitil covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oUgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturaUy occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein candesartan cilexitil is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW057P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein candesartan cilexitil is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing candesartan cilexitil from said composition in a pH-dependent manner.

19. A method for protecting candesartan cilexitil from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of candesartan cilexitU from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching candesartan cilexitil to said polypeptide.

21. A method for delivering candesartan cilexitil to a patient comprising administering to said patient a composition comprising: a polypeptide; and candesartan cilexitil covalently attached to said polypeptide.

22. The method of claim 21 wherein candesartan cilexitil is released from said composition by an enzyme-catalyzed release.

18 CW057P

23. The method of claim 21 wherein candesartan cilexitil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition fiirther comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and candesartan cilexitil covalently attached to the polypeptide. Also provided is a method for delivery of candesartan cilexitil to a patient comprising administering to the patient a composition comprising a polypeptide and candesartan cilexitil covalently attached to the polypeptide. Also provided is a method for protecting candesartan cilexitil from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of candesartan cilexitil from a composition comprising covalently attaching it to the polypeptide.

19 CW058P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CANDOXATRIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to candoxatril, as well as methods for protecting and administering candoxatril. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Candoxatril is a known pharmaceutical agent that is used in the treatment of heart failure and hypertension. Its chemical name is 4-[[[l-[3-[(2,3-dihydro-lH-inden-5- yl)oxy]-2-[(2-methoxyethoxy)methyl]-3-oxopropyl]cyclopentyl]carbonyl]amino]- cyclohexanecarboxylic acid. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical CW058P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabUity are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an oraUy administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW058P

shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug deUvery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW058P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (candoxatril) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching candoxatril to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising candoxatril microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and candoxatril covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW058P

Candoxatril preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The •microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting candoxatril from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering candoxatril to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, candoxatril is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, candoxatril is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and candoxatril is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, candoxatril is CW058P

released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, candoxatril is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching candoxatril to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, candoxatril and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW058P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Apphcation Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize candoxatril and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of candoxatril. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Candoxatril is the subject of EP 274234 B (1991), herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises candoxatril covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and poljφeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture. CW058P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW058P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophtiicity of the polypeptide.

Ionizing amino acids can be selected for pH confroUed peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but wUl ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW058P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug deUvery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly Umited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table Usts the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW058P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent deUvery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxyUc acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW058P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, candoxatril is covalently attached to the polypeptide via the carboxylic acid.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW058P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oUgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-candoxatril conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW058P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW058P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystaUized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be freated with diisopropylcarbodumide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW058P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW058P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and candoxatril covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein candoxatril is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW058P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein candoxatril is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing candoxatril from said composition in a pH-dependent manner.

19. A method for protecting candoxatril from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of candoxatril from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching candoxatril to said polypeptide.

21. A method for delivering candoxatril to a patient comprising administering to said patient a composition comprising: a polypeptide; and candoxatril covalently attached to said polypeptide.

22. The method of claim 21 wherein candoxatril is released from said composition by an enzyme-catalyzed release.

18 CW058P

23. The method of claim 21 wherein candoxatril is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and candoxatril covalently attached to the polypeptide. Also provided is a method for delivery of candoxatril to a patient comprising administering to the patient a composition comprising a polypeptide and candoxatril covalently attached to the polypeptide. Also provided is a method for protecting candoxatril from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of candoxatril from a composition comprising covalently attaching it to the polypeptide.

19 CW059P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CAPECITABINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to capecitabine, as well as methods for protecting and administering capecitabine. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Capecitabine is a known pharmaceutical agent that is used in the treatment of colorectal cancer. Its chemical name is pentyl l-(5-deoxy-.beta.-D-ribofuranosyl)-5- fluoro-l,2-dihydro-2-oxo-4-pyrimidinecarbamate. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW059P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compUance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across Upid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabUity of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW059P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW059P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labUe active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is Umited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (capecitabine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching capecitabine to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising capecitabine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and capecitabine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Capecitabine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW059P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting capecitabine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering capecitabine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, capecitabine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, capecitabine is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and capecitabine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, capecitabine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, capecitabine is released from the CW059P

composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching capecitabine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, capecitabine and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW059P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize capecitabine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of capecitabine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Capecitabine is the subject of U.S. Patent Numbers 4,966,891 and 5,472,949, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises capecitabine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW059P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oU drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW059P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controUed peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW059P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. GeneraUy, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly hmited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW059P

deUvery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW059P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, capecitabine is covalently attached to the polypeptide via the hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW059P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophUic compounds are absorbed through the intestinal epitheUa efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-capecitabine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW059P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

AIcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be freated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW059P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystaUized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW059P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and capecitabine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein capecitabine is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein capecitabine is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing capecitabine from said composition in a pH-dependent manner.

19. A method for protecting capecitabine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of capecitabine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching capecitabine to said polypeptide.

21. A method for delivering capecitabine to a patient comprising administering to said patient a composition comprising: a polypeptide; and capecitabine covalently attached to said polypeptide.

22. The method of claim 21 wherein capecitabine is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein capecitabine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

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Abstract

A composition comprising a polypeptide and capecitabine covalently attached to the polypeptide. Also provided is a method for delivery of capecitabine to a patient comprising administering to the patient a composition comprising a polypeptide and capecitabine covalently attached to the polypeptide. Also provided is a method for protecting capecitabine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of capecitabine from a composition comprising covalently attaching it to the polypeptide.

19 CW060P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CAPTOPRIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a poljφeptide that is preferably covalently attached to captopril, as well as methods for protecting and administering captopril. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Captopril is a known pharmaceutical agent that is used in the treatment of hypertension. Its chemical name is l-(3-mercapto-2-methyl-l-oxopropyl)-L-proline. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW060P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique, increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibiUty and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incoφoratiήg adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW060P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and Unking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW060P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (captopril) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching captopril to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising captopril microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and captopril covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturaUy occurring amino acids, (iii) a heteropolymer of two or more naturaUy occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Captopril preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW060P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-teiminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting captopril from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering captopril to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the poljφeptide. In a prefeoed embodiment, captopril is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, captopril is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and captopril is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, captopril is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, captopril is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW060P

confroUed by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching captopril to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, captopril and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutaπuc acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW060P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize captopril and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of captopril. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Captopril is the subject of U.S. Patent Number5 ,238,924, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises captopril covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturaUy occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW060P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overaU stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW060P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW060P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW060P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oUgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyrogfutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW060P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the ahmentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, captopril is covalentiy attached to the polypeptide via the carboxylic acid.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW060P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the fransported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facUitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-captopril conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW060P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be freated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW060P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW060P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and captopril covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein captopril is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW060P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein captopril is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing captopril from said composition in a pH-dependent manner.

19. A method for protecting captopril from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of captopril from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching captopril to said polypeptide.

21. A method for delivering captopril to a patient comprising administering to said patient a composition comprising: a polypeptide; and captopril covalently attached to said polypeptide.

22. The method of claim 21 wherein captopril is released from said composition by an enzyme-catalyzed release.

17 CW060P

23. The method of claim 21 wherein captopril is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW060P

Abstract

A composition comprising a polypeptide and captopril covalentiy attached to the polypeptide. Also provided is a method for delivery of captopril to a patient comprising administering to the patient a composition comprising a polypeptide and captopril covalently attached to the polypeptide. Also provided is a method for protecting captopril from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of captopril from a composition comprising covalentiy attaching it to the polypeptide.

19 CW061P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING

CARBAMAZEPINE

FTELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to carbamazepine, as well as methods for protecting and administering carbamazepine. This novel compound, refeoed to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Carbamazepine is a known pharmaceutical agent that is used in the treatment of epilepsy. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical CW061P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered oraUy in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabUity of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabUity of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW061P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW061P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is Umited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (carbamazepine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching carbamazepine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oUgopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide wiU stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising carbamazepine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and carbamazepine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids. CW061P

Carbamazepine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the poljφeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting carbamazepine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering carbamazepine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, carbamazepine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, carbamazepine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and carbamazepine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed CW061P

embodiment, carbamazepine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, carbamazepine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching carbamazepine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, carbamazepine and a second active agent can be copolymerized in step (c). In ,another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW061P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize carbamazepine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of carbamazepine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Carbamazepine is the subject of U.S. Patent Numbers 5,284,662 and Re. 34,990, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises carbamazepine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (u) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracmre. CW061P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stabiUty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW0 1P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stabiUty of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide wUl be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH confroUed peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can aU be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW061P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. GeneraUy, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW061P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-teiminus, the C-teπninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchlorofoπnate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW061P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not hmited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, carbamazepine is covalently attached to the polypeptide via the amido group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW061P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epitheha efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-carbamazepine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW061P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AcidVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW061P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodumide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alky 1 Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW061P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW061P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and carbamazepine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein carbamazepine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW061P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein carbamazepine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing carbamazepine from said composition in a pH-dependent manner.

19. A method for protecting carbamazepine from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of carbamazepine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching carbamazepine to said polypeptide.

21. A method for delivering carbamazepine to a patient comprising administering to said patient a composition comprising: a polypeptide; and carbamazepine covalently attached to said polypeptide.

22. The method of claim 21 wherein carbamazepine is released from said composition by an enzyme-catalyzed release.

18 CW061P

23. The method of claim 21 wherein carbamazepine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and carbamazepine covalently attached to the polypeptide. Also provided is a method for delivery of carbamazepine to a patient comprising administering to the patient a composition comprising a polypeptide and carbamazepine covalently attached to the polypeptide. Also provided is a method for protecting carbamazepine from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of carbamazepine from a composition comprising covalently attaching it to the polypeptide.

19 CW062P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CARBIDOPA AND LEVODOPA

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to carbidopa and levodopa, as well as methods for protecting and administering carbidopa and levodopa. This novel compound, refened to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Carbidopa and levodopa are known pharmaceutical agents that are used together in the treatment of Parkinson's disease. Each is commercially available and readily manufactured using published synthetic schemes by those of ordinary skiU in the art. Carbidopa's stracmre is:

Levodopa' s structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability CW062P

of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective deUvery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in Ueu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified CW062P

amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stabUity and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on (Uffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibUity. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres sweU by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that CW062P

incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexfran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (carbidopa and levodopa) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching carbidopa and levodopa to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, deUvery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising carbidopa and levodopa microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and carbidopa and levodopa covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, CW062P

(iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Carbidopa and levodopa preferably are covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-teπninus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the poljφeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting carbidopa and levodopa from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering carbidopa and levodopa to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, carbidopa and levodopa is CW062P

released from the composition by an enzyme-catalyzed release. In another prefened embodiment, carbidopa and levodopa is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and carbidopa and levodopa is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, carbidopa and levodopa is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, carbidopa and levodopa is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching carbidopa and levodopa to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, carbidopa and levodopa and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an CW062P

ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize carbidopa and levodopa and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of carbidopa and levodopa. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted deUvery of active agents to specifics sites of action.

The composition of the invention comprises carbidopa and levodopa covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The CW062P

folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stabiUty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and CW062P

at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stabiUty of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the poljφeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain CW062P

length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily confroUed by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug deUvery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW062P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

11 CW062P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pjrroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW062P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, carbidopa and levodopa are each covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a soUd state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asjαnmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW062P

mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-carbidopa and levodopa conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

14 CW062P

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopjrridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW062P

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the detaUs shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW062P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and carbidopa and levodopa covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturaUy occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein carbidopa and levodopa is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW062P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein carbidopa and levodopa is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said poljφeptide is capable of releasing carbidopa and levodopa from said composition in a pH-dependent manner.

19. A method for protecting carbidopa and levodopa from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of carbidopa and levodopa from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching carbidopa and levodopa to said polypeptide.

21. A method for delivering carbidopa and levodopa to a patient comprising administering to said patient a composition comprising: a polypeptide; and carbidopa and levodopa covalently attached to said polypeptide.

22. The method of claim 21 wherein carbidopa and levodopa is released from said composition by an enzyme-catalyzed release.

18 CW062P

23. The method of claim 21 wherein carbidopa and levodopa is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and carbidopa and levodopa covalently attached to the polypeptide. Also provided is a method for deUvery of carbidopa and levodopa to a patient comprising administering to the patient a composition comprising a poljφeptide and carbidopa and levodopa covalently attached to the polypeptide. Also provided is a method for protecting carbidopa and levodopa from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of carbidopa and levodopa from a composition comprising covalentiy attaching it to the polypeptide.

19 CW063P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CARBOPLATIN

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to carboplatin, as well as methods for protecting and administering carboplatin. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Carboplatin is a known pharmaceutical agent that is used in the treatment of ovarian cancer. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical CW063P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabiUty are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf Ufe or survival in the stomach, wiU assure dosage reproducibUity and perhaps even reduce the number of dosages required which could improve patient compUance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabUity of cellular membranes and fransport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW063P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag deUvery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW063P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (carboplatin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching carboplatin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabUize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzjrcnatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising carboplatin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and carboplatin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids. CW063P

Carboplatin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the poljφeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting carboplatin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering carboplatin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefened embodiment, carboplatin is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, carboplatin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and carboplatin is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, carboplatin is CW063P

released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, carboplatin is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching carboplatin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, carboplatin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW063P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize carboplatin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of carboplatin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises carboplatin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW063P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, coniϊgurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are estabUshed during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW063P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier poljφeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the poljφeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW063P

active agent release is primarily controlled by the enzj atic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug deUvery. Generally, it was only possible to load up to 1/10 of the total drug-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW063P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oUgopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly fooned free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW063P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutaπuc acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enz rmes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-teπninus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, carboplatin is covalently attached to the polypeptide via the amine groups.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW063P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facihtate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-carboplatin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW063P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with dusopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW063P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystalUzed from hot water.

γ-Alkyl Glutamate C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW063P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and carboplatin covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oUgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein carboplatin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW063P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein carboplatin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing carboplatin from said composition in a pH-dependent manner.

19. A method for protecting carboplatin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of carboplatin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching carboplatin to said polypeptide.

21. A method for delivering carboplatin to a patient comprising administering to said patient a composition comprising: a polypeptide; and carboplatin covalently attached to said polypeptide.

22. The method of claim 21 wherein carboplatin is released from said composition by an enzyme-catalyzed release.

17 CW063P

23. The method of claim 21 wherein carboplatin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW063P

Abstract

A composition comprising a polypeptide and carboplatin covalently attached to the polypeptide. Also provided is a method for delivery of carboplatin to a patient comprising administering to the patient a composition comprising a polypeptide and carboplatin covalentiy attached to the polypeptide. Also provided is a method for protecting carboplatin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of carboplatin from a composition comprising covalently attaching it to the polypeptide.

19 CW064P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING

CARISOPRODOL

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to carisoprodol, as well as methods for protecting and administering carisoprodol. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combimng it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Carisoprodol is a known phaonaceutical agent that is used in the treatment of skeletal muscle spasm. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW064P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabUity of cellular membranes and fransport across lipid bUayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabUity of ceUular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW064P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug deUvery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW064P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (carisoprodol) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching carisoprodol to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising carisoprodol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and carisoprodol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturaUy occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Carisoprodol preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW064P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting carisoprodol from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering carisoprodol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefeoed embodiment, carisoprodol is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, carisoprodol is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and carisoprodol is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, carisoprodol is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, carisoprodol is released from the CW064P

composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching carisoprodol to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, carisoprodol and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW064P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize carisoprodol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of carisoprodol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises carisoprodol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW064P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overaU stabUity of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW064P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW064P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus apphcation, for those active agents attached to pendant

10 CW064P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW064P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, carisoprodol is covalently attached to the polypeptide via the amido group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW064P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-carisoprodol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW064P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodumide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW064P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW064P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and carisoprodol covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein carisoprodol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein carisoprodol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing carisoprodol from said composition in a pH-dependent manner.

19. A method for protecting carisoprodol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of carisoprodol from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching carisoprodol to said polypeptide.

21. A method for delivering carisoprodol to a patient comprising administering to said patient a composition comprising: a polypeptide; and carisoprodol covalently attached to said polypeptide.

22. The method of claim 21 wherein carisoprodol is released from said composition by an enzyme-catalyzed release.

17 CW064P

23. The method of claim 21 wherein carisoprodol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW064P

Abstract

A composition comprising a polypeptide and carisoprodol covalently attached to the polypeptide. Also provided is a method for delivery of carisoprodol to a patient comprising administering to the patient a composition comprising a polypeptide and carisoprodol covalentiy attached to the polypeptide. Also provided is a method for protecting carisoprodol from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of carisoprodol from a composition comprising covalently attaching it to the polypeptide.

19 CW065P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CARVEDILOL

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to carvedilol, as well as methods for protecting and administering carvedilol. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INNENTION

Carvedilol is a known pharmaceutical agent that is used in the treatment of heart failure. Its chemical name is l-(9H-carbazol-4-yloxy)-3-[[2-(2-methoxyphenoxy) ethyl]amino]-2-propanol. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW065P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings, incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW065P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag deUvery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: Unking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system.* Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW065P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(carvedilol) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching carvedilol to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising carvedilol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and carvedilol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Carvedilol preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the poljφeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW065P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting carvedilol from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering carvedilol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a poljφeptide and an active agent covalentiy attached to the polypeptide. In a prefened embodiment, carvedilol is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, carvedilol is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and carvedilol is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, carvedilol is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, carvedilol is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW065P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching carvedilol to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, carvedilol and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW065P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize carvedilol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of carvedilol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Carvedilol is the subject of U.S. Patent Numbers 4,503,067, 5,760,069, and 5,902,821, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises carvedilol covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW065P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stabUity, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW065P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the canier peptide and the active agent. CW065P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW065P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW065P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, carvedilol is covalently attached to the polypeptide via the hydrozyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW065P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-carvedilol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW065P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with (UisopropylcarbooUimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW065P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW065P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and carvedilol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein carvedilol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW065P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein carvedilol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing carvedilol from said composition in a pH-dependent manner.

19. A method for protecting carvedilol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of carvedilol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching carvedilol to said polypeptide.

21. A method for delivering carvedilol to a patient comprising administering to said patient a composition comprising: a polypeptide; and carvedilol covalently attached to said polypeptide.

22. The method of claim 21 wherein carvedilol is released from said composition by an enzyme-catalyzed release.

17 CW065P

23. The method of claim 21 wherein carvedilol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW065P

Abstract

A composition comprising a polypeptide and carvedilol covalently attached to the polypeptide. Also provided is a method for delivery of carvedilol to a patient comprising administering to the patient a composition comprising a polypeptide and carvedilol covalentiy attached to the polypeptide. Also provided is a method for protecting carvedilol from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of carvedilol from a composition comprising covalently attaching it to the polypeptide.

19 CW066P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CASPOFUNGIN

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to caspofiingin, as well as methods for protecting and administering caspofiingin. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Caspofiingin is a known pharmaceutical agent that is used in the treatment of bacterial and fungal infections. Its chemical name is l-[(4R,5S)-5-[(2- aminoethyl)amino]-N2-( 10, 12-dimethyl- 1 -oxotetradecyl)-4-hydroxy-L-omithine] -5- [(3R)-3-hydroxy-L-ornithine]pneumocandin B0. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered CW066P

product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations. CW066P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drug conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW066P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (caspofungin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching caspofungin to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising caspofungin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and caspofungin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW066P

heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Caspofungin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the poljφeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting caspofungin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering caspofungin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, caspofungin is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, caspofungin is released in a time-dependent manner based on the pharmacokinetics of the enzyme- CW066P

catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and caspofungin is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, caspofungin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, caspofungin is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching caspofungin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, caspofungin and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the CW066P

glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize caspofungin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of caspofungin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Caspofungin is the subject of WO 94/21677 (1994), based on U.S. Patent

Application Number 32847 (1993), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises caspofungin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The CW066P

folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and CW066P

at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipopbilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophihcity of the poljφeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain CW066P

length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW066P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oUgopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

11 CW066P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This canier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW066P

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of Unking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, caspofungin is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW066P

mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-caspofungin conjugate is formulated into a tablet using suitable excipients and.can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to

0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide canier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

14 CW066P

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW066P

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although Ulustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW066P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and caspofungin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein caspofungin is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW066P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein caspofungin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing caspofungin from said composition in a pH-dependent manner.

19. A method for protecting caspofungin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of caspofungin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching caspofungin to said polypeptide.

21. A method for delivering caspofungin to a patient comprising administering to said patient a composition comprising: a polypeptide; and caspofungin covalently attached to said polypeptide.

22. The method of claim 21 wherein caspofungin is released from said composition by an enzyme-catalyzed release.

18 CW066P

23. The method of claim 21 wherein caspofungin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and caspofungin covalently attached to the polypeptide. Also provided is a method for delivery of caspofungin to a patient comprising administering to the patient a composition comprising a polypeptide and caspofungin covalently attached to the polypeptide. Also provided is a method for protecting caspofungin from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of caspofungin from a composition comprising covalently attaching it to the polypeptide.

19 CW067P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFACLOR

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to cefaclor, as well as methods for protecting and administering cefaclor. This novel compound, refened to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefaclor is a known phaimaceutical agent that is used in the treatment of bronchitis. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW067P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compUance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabUity of cellular membranes and transport across lipid bUayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW067P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW067P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cefaclor) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalentiy attaching cefaclor to the N-teπninus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefaclor microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefaclor covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cefaclor preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW067P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terπύnus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefaclor from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefaclor to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cefaclor is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cefaclor is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and cefaclor is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, cefaclor is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, cefaclor is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW067P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cefaclor to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefaclor and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW067P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cefaclor and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefaclor. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cefaclor covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturaUy occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW067P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be CW067P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the poljφeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW067P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with. the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

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The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW067P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefaclor is covalentiy attached to the polypeptide via the carboxylic acid.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

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There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefaclor conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

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precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW067P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW067P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and cefaclor covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefaclor is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceuticaUy acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefaclor is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefaclor from said composition in a pH-dependent manner.

19. A method for protecting cefaclor from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cefaclor from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cefaclor to said polypeptide.

21. A method for delivering cefaclor to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefaclor covalently attached to said polypeptide.

22. The method of claim 21 wherein cefaclor is released from said composition by an enzyme-catalyzed release.

17 CW067P

23. The method of claim 21 wherein cefaclor is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

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Abstract

A composition comprising a polypeptide and cefaclor covalently attached to the polypeptide. Also provided is a method for delivery of cefaclor to a patient comprising administering to the patient a composition comprising a polypeptide and cefaclor covalently attached to the polypeptide. Also provided is a method for protecting cefaclor from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cefaclor from a composition comprising covalentiy attaching it to the polypeptide.

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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFADROXIL

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cefadroxil, as well as methods for protecting and administering cefadroxil. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both weU studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefadroxil is a known pharmaceutical agent that is used in the treatment of bacterial infections. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW068P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stabUity of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and fransport across lipid bUayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent deUvery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW068P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW068P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(cefadroxil) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching cefadroxil to the N- ter inus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefadroxil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefadroxil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Cefadroxil preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW068P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefadroxil from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefadroxil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, cefadroxil is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cefadroxil is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cefadroxil is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cefadroxil is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cefadroxil is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW068P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching cefadroxil to a side chain of an amino acid to form an active agent/aπ no acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefadroxil and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW068P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cefadroxil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefadroxil. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cefadroxil covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturaUy occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW068P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be CW068P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but wUl ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can aU be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW068P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW068P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teiminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

11 CW068P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefadroxil is covalentiy attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW068P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefadroxil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW068P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylaπύne or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW068P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW068P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cefadroxil covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefadroxil is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW068P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefadroxil is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefadroxil from said composition in a pH-dependent manner.

19. A method for protecting cefadroxil from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cefadroxil from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cefadroxil to said polypeptide.

21. A method for delivering cefadroxil to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefadroxil covalently attached to said polypeptide.

22. The method of claim 21 wherein cefadroxil is released from said composition by an enzyme-catalyzed release.

17 CW068P

23. The method of claim 21 wherein cefadroxil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW068P

Abstract

A composition comprising a polypeptide and cefadroxil covalently attached to the polypeptide. Also provided is a method for delivery of cefadroxU to a patient comprising administering to the patient a composition comprising a polypeptide and cefadroxil covalentiy attached to the polypeptide. Also provided is a method for protecting cefadroxil from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cefadroxil from a composition comprising covalently attaching it to the polypeptide.

19 CW069P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFAZOLIN

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cefazohn, as well as methods for protecting and administering cefazohn. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefazohn is a known pharmaceutical agent that is used in the treatment of respiratory tract infections, urinary tract infections, skin and skin structure infections, biliary fract infections, bone and joint infections, genital infections, septicemia, and endocarditis caused by susceptible bacteria. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW069P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an oraUy administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and fransport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stabihty and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW069P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreUable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typicaUy require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug deUvery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW069P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cefazohn) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching cefazohn to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain appUcations, the polypeptide wiU stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising cefazohn microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefazohn covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cefazohn preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW069P

carboxylic acid and is covalentiy attached to the N-teoninus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefazohn from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefazohn to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cefazohn is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cefazohn is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cefazohn is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cefazohn is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cefazohn is released from the composition in a sustained release. CW069P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cefazolin to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefazolin and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutaπuc acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW069P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize cefazolin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefazolin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cefazolin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW069P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW069P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide wiU be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophiUcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW069P

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the Ueum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW069P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit foπning a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW069P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enz ies in the alimentary fract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefazohn is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW069P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system wiU involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported substrate. They include the amino acid, oUgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefazolin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW069P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-teoninus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW069P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW069P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and cefazolin covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefazolin is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW069P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefazolin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefazolin from said composition in a pH-dependent manner.

19. A method for protecting cefazolin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for confrolUng release of cefazolin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cefazolin to said polypeptide.

21. A method for delivering cefazolin to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefazolin covalently attached to said polypeptide.

22. The method of claim 21 wherein cefazolin is released from said composition by an enzyme-catalyzed release.

17 CW069P

23. The method of claim 21 wherein cefazolin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW069P

Abstract

A composition comprising a polypeptide and cefazolin covalentiy attached to the polypeptide. Also provided is a method for delivery of cefazolin to a patient comprising administering to the patient a composition comprising a polypeptide and cefazohn covalently attached to the polypeptide. Also provided is a method for protecting cefazolin from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controUing release of cefazolin from a composition comprising covalently attaching it to the polypeptide.

19 CW070P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFDINIR

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cefdinir, as well as methods for protecting and administering cefdinir. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefdinir is a known phaimaceutical agent that is used in the treatment of acute maxillary sinusitis, acute exacerbations of chronic bronchitis, pharyngitis, tonsilitis, community-acquired pneumonia and bacterial skin infections. Its chemical name is [6R- [6alpha,7beta(Z)]]-7-[[(2-amino-4-thiazolyl)(hydroxjάιmno) acetyl]amino]-3-ethenyl-8- oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW070P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibUity and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabiUty of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW070P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW070P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR apphcation, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cefdinir) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching cefdinir to the N-teπninus, the C-terminus or directly to the amino acid side chain of an oUgopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controUed, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefdinir microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefdinir covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cefdinir preferably is covalently attached to a side chain, the N-terminus or the C- terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic CW070P

acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalentiy attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefdinir from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefdinir to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, cefdinir is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cefdinir is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cefdinir is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cefdinir is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cefdinir is released from the composition in a sustained release. In yet CW070P

another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching cefdinir to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefdinir and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW070P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cefdinir and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefdinir. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cefdinir is the subject of EP 105459 B (1989), based on US Application Serial

Number 428,970 (1982) and EP 304019 B (1995), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cefdinir covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturaUy occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW070P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW070P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW070P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW070P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW070P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is teπninated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not Umited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefdinir is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW070P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefdinir conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW070P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW070P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW070P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and cefdinir covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefdinir is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW070P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefdinir is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefdinir from said composition in a pH-dependent manner.

19. A method for protecting cefdinir from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of cefdinir from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cefdinir to said polypeptide.

21. A method for delivering cefdinir to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefdinir covalently attached to said polypeptide.

22. The method of claim 21 wherein cefdinir is released from said composition by an enzyme-catalyzed release.

17 CW070P

23. The method of claim 21 wherein cefdinir is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW070P

Abstract

A composition comprising a polypeptide and cefdinir covalentiy attached to the polypeptide. Also provided is a method for delivery of cefdinir to a patient comprising administering to the patient a composition comprising a polypeptide and cefdinir covalently attached to the polypeptide. Also provided is a method for protecting cefdinir from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of cefdinir from a composition comprising covalentiy attaching it to the polypeptide.

19 CW071P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFIXIME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to cefixime, as well as methods for protecting and administering cefixime. This novel compound, referred to as a CARRJERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefixime is a known pharmaceutical agent that is used in the treatment of respiratory tract infections, gononhea, biliary tract infection and pediatric otitis media. Its chemical name is [6R-[6alpha,7beta(Z)]]-7-[[(2-amino-4-thiazolyl)[(carboxymethoxy) imino]acetyl]amino]-3-ethenyl-8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW071P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibiUty and perhaps even reduce the number of dosages required which could improve patient compUance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW071P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typicaUy require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW071P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brash-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cefixime) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalentiy attaching cefixime to the N-terminus, the C-teoninus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefixime microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefixime covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW071P

Cefixime preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefixime from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefixime to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, cefixime is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cefixime is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cefixime is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cefixime is released CW071P

from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, cefixime is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is confroUed by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of: (a) attaching cefixime to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefixime and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutaπuc acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW071P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize cefixime and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefixime. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cefixime is the subject of EP 30360 B (1987), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cefixime covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW071P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stabiUty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW071P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the poljφeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW071P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW071P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

11 CW071P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytjrrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefixime is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW071P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophUic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefixime conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW071P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW071P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW071P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and cefixime covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefixime is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW071P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefixime is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefixime from said composition in a pH-dependent manner.

19. A method for protecting cefixime from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cefixime from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cefixime to said polypeptide.

21. A method for delivering cefixime to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefixime covalently attached to said polypeptide.

22. The method of claim 21 wherein cefixime is released from said composition by an enzyme-catalyzed release.

17 CW071P

23. The method of claim 21 wherein cefixime is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW071P

Abstract

A composition comprising a polypeptide and cefixime covalently attached to the polypeptide. Also provided is a method for delivery of cefixime to a patient comprising administering to the patient a composition comprising a polypeptide and cefixime covalently attached to the polypeptide. Also provided is a method for protecting cefixime from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cefixime from a composition comprising covalently attaching it to the polypeptide.

19 CW072P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING NORADRENALIN AND DOPAMINE REUPTAKE INHIBITOR

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to the noradrenalin and dopamine reuptake inhibitor, as well as methods for protecting and administering the noradrenalin and dopamine reuptake inhibitor. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

The noradrenalin and dopamine reuptake inhibitor of the present invention is used in the treatment of attention deficit hyperactivity disorder (ADHA). Its chemical name is [2S-(2alpha,3alpha,5alpha)]-2-(3,5-difluorophenyl)-3,5-dimethyl-2-moφholinol hydrochloride. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor. CW072P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW072P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of poljφeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW072P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (noradrenalin and dopamine reuptake inhibitor) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching the noradrenalin and dopamine reuptake inhibitor to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising the noradrenalin and dopamine reuptake inhibitor microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and the noradrenalin and dopamine reuptake inhibitor covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW072P

The noradrenalin and dopamine reuptake inhibitor preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the poljφeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C- terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting the noradrenalin and dopamine reuptake inhibitor from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering the noradrenalin and dopamine reuptake inhibitor to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, the noradrenalin and dopamine reuptake inhibitor is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, the noradrenalin and dopamine reuptake inhibitor is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the CW072P

composition further comprises a microencapsulating agent and the noradrenalin and dopamine reuptake inhibitor is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, the noradrenalin and dopamine reuptake inhibitor is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, the noradrenahn and dopamine reuptake inhibitor is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching the noradrenalin and dopamine reuptake inhibitor to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, the noradrenalin and dopamine reuptake inhibitor and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant CW072P

group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize the noradrenalin and dopamine reuptake inhibitor and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of the noradrenalin and dopamine reuptake inhibitor. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The noradrenalin and dopamine reuptake inhibitor of the present invention is the subject of EP 426416 B (1994), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises the noradrenalin and dopamine reuptake inhibitor covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and CW072P

turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more hke a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is CW072P

a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired. CW072P

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW072P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a poljφeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

π CW072P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytjTosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW072P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the noradrenalin and dopamine reuptake inhibitor is covalently attached to the polypeptide via the hydroxyl.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW072P

mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-noradrenaUn and dopamine reuptake inhibitor conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

14 CW072P

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyπolidinopjridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-AIkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW072P

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AlkyI Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be hmited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW072P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and noradrenalin and dopamine reuptake inhibitor covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein the noradrenalin and dopamine reuptake inhibitor is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

17 CW072P

11. The composition of claim 1 further comprising an adjuvant.

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein the noradrenalin and dopamine reuptake inhibitor is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing the noradrenalin and dopamine reuptake inhibitor from said composition in a pH- dependent manner.

19. A method for protecting the noradrenalin and dopamine reuptake inhibitor from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of the noradrenalin and dopamine reuptake inhibitor from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching the noradrenalin and dopamine reuptake inhibitor to said polypeptide.

21. A method for delivering the noradrenalin and dopamine reuptake inhibitor to a patient comprising administering to said patient a composition comprising:

18 CW072P

a polypeptide; and the noradrenalin and dopamine reuptake inhibitor covalently attached to said polypeptide.

22. The method of claim 21 wherein the noradrenalin and dopamine reuptake inhibitor is released from said composition by an enzyme-catalyzed release.

23. The method of claim 21 wherein the noradrenalin and dopamine reuptake inhibitor is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and the noradrenalin and dopamine reuptake inhibitor covalently attached to the polypeptide. Also provided is a method for delivery of the noradrenalin and dopamine reuptake inhibitor to a patient comprising administering to the patient a composition comprising a polypeptide and the noradrenalin and dopamine reuptake inhibitor covalently attached to the polypeptide. Also provided is a method for protecting the noradrenalin and dopamine reuptake inhibitor from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controUing release of the noradrenalin and dopamine reuptake inhibitor from a composition comprising covalently attaching it to the polypeptide.

19 CW073P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFOTAXIME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to cefotaxime, as well as methods for protecting and administering cefotaxime. This novel compound, refeoed to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefotaxime is a known pharmaceutical agent that is used in the treatment of serious bone and joint infections, serious intra-abdominal and gynecologic infections (including peritonitis, endometritis, pelvic inflammatory disease, pelvic cellulitis), meningitis and other CNS infections, serious lower respiratory fract infections (including pneumonia), bacteremia/septicemia, serious skin and skin structure infections, and serious urinary fract infections caused by susceptible bacteria. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its chemical name is (6R,7R)-7-[2-(2-Amino-4-thiazolyl) glyoxylamido]-8-oxo-3-vinyl-5-thia- 1 -azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7²(Z)-[O-(carboxymethyl)oxime].

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW073P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compUance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeabUity of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stabihty and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW073P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight earners are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW073P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cefotaxime) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cefotaxime to the N-terminus, the C-teoninus or directly to the amino acid side chain of an oligopeptide or poljφeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is confroUed, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefotaxime microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefotaxime covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Cefotaxime preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW073P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefotaxime from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefotaxime to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cefotaxime is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cefotaxime is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and cefotaxime is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, cefotaxime is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, cefotaxime is released from the composition in a CW073P

sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cefotaxime to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefotaxime and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW073P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cefotaxime and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefotaxime. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cefotaxime covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW073P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW073P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophtiicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW073P

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW073P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teπninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW073P

maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-teoninus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefotaxime is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW073P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier poljφeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefotaxime conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW073P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW073P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW073P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and cefotaxime covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefotaxime is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW073P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefotaxime is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefotaxime from said composition in a pH-dependent manner.

19. A method for protecting cefotaxime from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cefotaxime from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cefotaxime to said polypeptide.

21. A method for delivering cefotaxime to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefotaxime covalently attached to said polypeptide.

22. The method of claim 21 wherein cefotaxime is released from said composition by an enzyme-catalyzed release.

17 CW073P

23. The method of claim 21 wherein cefotaxime is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW073P

Abstract

A composition comprising a polypeptide and cefotaxime covalentiy attached to the polypeptide. Also provided is a method for delivery of cefotaxime to a patient comprising administering to the patient a composition comprising a polypeptide and cefotaxime covalentiy attached to the polypeptide. Also provided is a method for protecting cefotaxime from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cefotaxime from a composition comprising covalently attaching it to the polypeptide.

19 CW074P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFOTETAN

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cefotetan, as well as methods for protecting and administering cefotetan. This novel compound, referred to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefotetan is a known pharmaceutical agent that is used in the treatment of septicemia, genitourinary, biliary and respiratory tract infections, and in postoperativewound infection prophylaxis. Its chemical name is [6R-(6alpha,7alpha)]-7- [[[4-(2-aπuno-l-carboxy-2-oxoethylidene)-l,3-ditWetan-2-yl]carbonyl]arnino]-7- methoxy-3-[[( 1 -methyl- 1 H-tettazol-5-yl)thio] methyl]-8-oxo-5-thia- 1 -azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW074P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibiUty and perhaps even reduce the number of dosages required which could improve patient compUance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW074P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW074P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cefotetan) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching cefotetan to the N-terminus, the C-teoninus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefotetan microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefotetan covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids. CW074P

Cefotetan preferably is covalently attached to a side chain, the N-teoninus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationaUy protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefotetan from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefotetan to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cefotetan is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cefotetan is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cefotetan is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cefotetan is released CW074P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cefotetan is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching cefotetan to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefotetan and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW074P

The general applications of this invention to other active phaimaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize cefotetan and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefotetan. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cefotetan is the subject of GB 1604739 (1981), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cefotetan covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Protein-? fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW074P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystaUine solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW074P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW074P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. GeneraUy, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW074P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the ohgopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW074P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefotetan is covalentiy attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW074P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefotetan conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW074P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW074P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AIkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW074P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and cefotetan covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefotetan is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW074P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefotetan is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefotetan from said composition in a pH-dependent manner.

19. A method for protecting cefotetan from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of cefotetan from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching cefotetan to said polypeptide.

21. A method for delivering cefotetan to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefotetan covalently attached to said polypeptide.

22. The method of claim 21 wherein cefotetan is released from said composition by an enzyme-catalyzed release.

17 CW074P

23. The method of claim 21 wherein cefotetan is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW074P

Abstract

A composition comprising a polypeptide and cefotetan covalentiy attached to the polypeptide. Also provided is a method for delivery of cefotetan to a patient comprising administering to the patient a composition comprising a polypeptide and cefotetan covalently attached to the polypeptide. Also provided is a method for protecting cefotetan from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cefotetan from a composition comprising covalently attaching it to the polypeptide.

19 CW075P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFOXITIN

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cefoxitin, as well as methods for protecting and administering cefoxitin. This novel compound, referred to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefoxitin is a known pharmaceutical agent that is used in the treatment of serious infections of the lower respiratory tract, skin and skin stracmre, bone and joint, and urinary ttact; septicemia; gynecologic infections (including endometritis, pelvic cellulitis, and pelvic inflammatory disease); and intta-abdominal infections (including peritonitis and intta-abdominal abscess) caused by susceptible bacteria. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW075P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid πucrospheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW075P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW075P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cefoxitin) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalentiy attaching cefoxitin to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefoxitin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefoxitin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids. CW075P

Cefoxitin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the poljφeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefoxitin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefoxitin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, cefoxitin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cefoxitin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cefoxitin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cefoxitin is released CW075P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cefoxitin is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching cefoxitin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefoxitin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW075P

The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cefoxitin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefoxitin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cefoxitin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the CW075P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW075P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW075P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 ' Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW075P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW075P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefoxitin is covalentiy attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW075P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefoxitin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW075P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terrninus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW075P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutaπuc acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW075P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cefoxitin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said poljφeptide is an ohgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefoxitin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW075P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefoxitin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefoxitin from said composition in a pH-dependent manner.

19. A method for protecting cefoxitin from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of cefoxitin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cefoxitin to said polypeptide.

21. A method for delivering cefoxitin to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefoxitin covalently attached to said polypeptide.

22. The method of claim 21 wherein cefoxitin is released from said composition by an enzyme-catalyzed release.

17 CW075P

23. The method of claim 21 wherein cefoxitin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW075P

Abstract

A composition comprising a polypeptide and cefoxitin covalentiy attached to the polypeptide. Also provided is a method for delivery of cefoxitin to a patient comprising administering to the patient a composition comprising a polypeptide and cefoxitin covalently attached to the polypeptide. Also provided is a method for protecting cefoxitin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cefoxitin from a composition comprising covalently attaching it to the polypeptide.

19 CW076P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFPODOXIME PROXETIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cefpodoxime proxetil, as well as methods for protecting and administering cefpodoxime proxetil. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefpodoxime proxetil is a known phaonaceutical agent that is used in the tteatment of mild to moderate infections of the upper and lower respiratory tract, skin and urinary ttact and sexually transmitted diseases. Its chemical name is [6R- [6alfa,7beta(Z)]]-7-[[(2-amino-4-thiazolyl)(methoxyimino)acetyl]amino]-3- (methoxymethyl)-8-oxo-5-thia- 1 -azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 1 -[[( 1 - methylethoxy)carbonyl]oxy]ethyl ester. Its stracture is:

\

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; CW076P

and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations. CW076P

Each of these technologies imparts enhanced stabihty and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW076P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cefpodoxime proxetil) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cefpodoxime proxetil to the N-terminus, the C-teraiinus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefpodoxime proxetil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefpodoxime proxetil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or CW076P

(vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cefpodoxime proxetil preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefpodoxime proxetil from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefpodoxime proxetil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, cefpodoxime proxetil is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cefpodoxime proxetil is released in a time-dependent manner based on the CW076P

pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cefpodoxime proxetil is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cefpodoxime proxetil is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefened embodiment, cefpodoxime proxetil is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a poljφeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cefpodoxime proxetil to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefpodoxime proxetil and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the CW076P

glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cefpodoxime proxetil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefpodoxime proxetil. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cefpodoxime proxetil is the subject of EP 49118 B (1986), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises cefpodoxime proxetil covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The CW076P

folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the theimodjrnamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and CW076P

at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain CW076P

length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily conttoUed by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug deUvery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW076P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin e (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a poljφeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

1 1 CW076P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enz πnatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW076P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of Unking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefpodoxime proxetil is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabiUzing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW076P

mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefpodoxime proxetil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

14 CW076P

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-AIkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW076P

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW076P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cefpodoxime proxetil covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefpodoxime proxetil is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW076P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefpodoxime proxetil is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefpodoxime proxetil from said composition in a pH-dependent manner.

19. A method for protecting cefpodoxime proxetil from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cefpodoxime proxetil from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching cefpodoxime proxetil to said polypeptide.

21. A method for delivering cefpodoxime proxetil to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefpodoxime proxetil covalently attached to said polypeptide.

22. The method of claim 21 wherein cefpodoxime proxetil is released from said composition by an enzyme-catalyzed release. CW076P

23. The method of claim 21 wherein cefpodoxime proxetil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and cefpodoxime proxetil covalentiy attached to the polypeptide. Also provided is a method for delivery of cefpodoxime proxetil to a patient comprising administering to the patient a composition comprising a polypeptide and cefpodoxime proxetil covalently attached to the polypeptide. Also provided is a method for protecting cefpodoxime proxetil from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cefpodoxime proxetil from a composition comprising covalently attaching it to the polypeptide.

19 CW077P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFPROZIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalentiy attached to cefprozil, as well as methods for protecting and administering cefprozil. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefprozil is a known phaonaceutical agent that is used in the treatment of upper respiratory ttact infections, otitis media, acute exacerbation of chronic bronchitis, and skin infections. Its chemical name is [6R-[6alpha,7beta(R*)]]-7-[[amino(4- hydroxyphenyl)acetyl]amino-8-oxo-3-( 1 -propenyl)-5-thia- 1 -azabicyclo[4.2.0]oct-2-ene- 2-carboxylic acid. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW077P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stabihty of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings, incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW077P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an ac.tive ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW077P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cefprozil) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalentiy attaching cefprozil to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefprozil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefprozil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cefprozil preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a CW077P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationaUy protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefprozil from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefprozil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, cefprozil is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cefprozil is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cefprozil is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cefprozil is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cefprozil is released from the composition in a sustained release. CW077P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cefprozil to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefprozil and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW077P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize cefprozil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefprozil. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cefprozil is the subject of GB 2135305 B (1987), based on US Patent Application

Serial Number 461833 (1983), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cefprozil covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturaUy occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary structure and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW077P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW077P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier poljφeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW077P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW077P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oUgopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW077P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acylttiazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefprozil is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW077P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefprozil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW077P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW077P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW077P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cefprozil covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefprozil is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW077P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefprozil is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefprozil from said composition in a pH-dependent manner.

19. A method for protecting cefprozil from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of cefprozil from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cefprozil to said polypeptide.

21. A method for delivering cefprozil to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefprozil covalently attached to said polypeptide.

22. The method of claim 21 wherein cefprozil is released from said composition by an enzyme-catalyzed release.

17 CW077P

23. The method of claim 21 wherein cefprozil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW077P

Abstract

A composition comprising a polypeptide and cefprozil covalentiy attached to the polypeptide. Also provided is a method for delivery of cefprozil to a patient comprising administering to the patient a composition comprising a polypeptide and cefprozil covalentiy attached to the polypeptide. Also provided is a method for protecting cefprozil from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controUing release of cefprozil from a composition comprising covalently attaching it to the polypeptide.

19 CW078P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFTAZIDIME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ceftazidime, as weU as methods for protecting and administering ceftazidime. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ceftazidime is a known phaonaceutical agent that is used in the treatment of bacterial infections. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW078P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW078P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight earners are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW078P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ceftazidime) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ceftazidime to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabiUze the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising ceftazidime microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ceftazidime covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Ceftazidime preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW078P

carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ceftazidime from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering ceftazidime to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, ceftazidime is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, ceftazidime is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and ceftazidime is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, ceftazidime is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, ceftazidime is released from the CW078P

composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ceftazidime to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ceftazidime and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW078P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ceftazidime and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ceftazidime. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted deUvery of active agents to specifics sites of action.

The composition of the invention comprises ceftazidime covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW078P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stabiUty of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW078P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the canier peptide and the active agent. CW078P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be conttoUed and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163. Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW078P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teiminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular ttansamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW078P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ceftazidime is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW078P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ceftazidime conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW078P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW078P

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW078P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ceftazidime covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ceftazidime is covalently attached to a side chain, the N-teoninus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW078P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ceftazidime is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ceftazidime from said composition in a pH-dependent manner.

19. A method for protecting ceftazidime from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ceftazidime from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching ceftazidime to said polypeptide.

21. A method for delivering ceftazidime to a patient comprising administering to said patient a composition comprising: a polypeptide; and ceftazidime covalently attached to said polypeptide.

22. The method of claim 21 wherein ceftazidime is released from said composition by an enzyme-catalyzed release.

17 CW078P

23. The method of claim 21 wherein ceftazidime is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW078P

Abstract

A composition comprising a polypeptide and ceftazidime covalently attached to the polypeptide. Also provided is a method for delivery of ceftazidime to a patient comprising administering to the patient a composition comprising a polypeptide and ceftazidime covalently attached to the polypeptide. Also provided is a method for protecting ceftazidime from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ceftazidime from a composition comprising covalently attaching it to the polypeptide.

19 CW079P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFTTBUTENAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ceftibuten, as well as methods for protecting and administering ceftibuten. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pha naceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ceftibuten is a known pharmaceutical agent that is used in the treatment of bacterial infections. Its chemical name is [6R-[6alpha,7beta(Z)]]-7-[[2-(2-amino-4- thiazolyl)-4-carboxy-l-oxo-2-butenyl]amino]-8-oxo 5-thia-l-azabicyclo[4.2.0]oct-2-ene- 2-ca rboxylic acid. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW079P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW079P

reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW079P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ceftibuten) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ceftibuten to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, deUvery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ceftibuten microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ceftibuten covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturaUy occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ceftibuten preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW079P

carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ceftibuten from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ceftibuten to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, ceftibuten is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, ceftibuten is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and ceftibuten is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, ceftibuten is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, ceftibuten is released from the composition in a sustained release. CW079P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching ceftibuten to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ceftibuten and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW079P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabiUze ceftibuten and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ceftibuten. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Ceftibuten is the subject of EP 136721 B (1993), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises ceftibuten covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropoljraπer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary structure and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW079P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stabUity is the solid reference state.

The major forces contributing to the thermodjαiamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stabUity, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overaU stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is hkely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW079P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophihcity is desired, then the carrier polypeptide wUl be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW079P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. GeneraUy, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be confroUed.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW079P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teπninus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-teiminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW079P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the poljφeptide using known techniques. Examples of Unking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ceftibuten is covalentiy attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW079P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ceftibuten conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW079P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with dusopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW079P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystaUized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW079P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ceftibuten covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturaUy occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ceftibuten is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW079P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceuticaUy acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ceftibuten is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ceftibuten from said composition in a pH-dependent manner.

19. A method for protecting ceftibuten from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of ceftibuten from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ceftibuten to said polypeptide.

21. A method for delivering ceftibuten to a patient comprising administering to said patient a composition comprising: a poljφeptide; and ceftibuten covalently attached to said polypeptide.

22. The method of claim 21 wherein ceftibuten is released from said composition by an enzyme-catalyzed release.

17 CW079P

23. The method of claim 21 wherein ceftibuten is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW079P

Abstract

A composition comprising a polypeptide and ceftibuten covalently attached to the polypeptide. Also provided is a method for delivery of ceftibuten to a patient comprising administering to the patient a composition comprising a polypeptide and ceftibuten covalently attached to the polypeptide. Also provided is a method for protecting ceftibuten from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ceftibuten from a composition comprising covalently attaching it to the polypeptide.

19 CW080P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING A BILE ACID TRANSPORT INHIBITOR AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to a bile acid transport inhibitor, as well as methods for protecting and administering a bile acid transport inhibitor. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

The bile acid ttansport inhibitor of the present invention is a known pharmaceutical agent that is used in the treatment of hypercholesterolemia. Its chemical name is (3R,5R)-rel-3-butyl-3-ehtyl-2,3,4,5-tetrahydro-7,8-dimethoxy-5-phenyl- 1 ,4- benzothiazepine 1,1 -dioxide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW080P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compUance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique, increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across Upid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bUe acids enhance permeabUity of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stabihty and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW080P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzjrmes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in CW080P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (The bile acid ttansport inhibitor of the present invention) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching the bile acid transport inhibitor of the present invention to the N- terminus, the C-terminus or directly to the amino acid side chain of an oUgopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain apphcations, the polypeptide will stabiUze the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising the bile acid ttansport inhibitor of the present invention microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and the bile acid transport inhibitor of the present invention covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW080P

The bile acid fransport inhibitor of the present invention preferably is covalentiy attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C- teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting the bile acid ttansport inhibitor of the present invention from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering the bile acid ttansport inhibitor of the present invention to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, the bile acid fransport inhibitor of the present invention is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, the bile acid ttansport inhibitor of the present invention is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the CW080P

composition further comprises a microencapsulating agent and the bile acid ttansport inhibitor of the present invention is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, the bile acid transport inhibitor of the present invention is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, the bUe acid transport inhibitor of the present invention is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching the bile acid transport inhibitor of the present invention to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, the bile acid transport inhibitor of the present invention and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a CW080P

pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabihze the bile acid ttansport inhibitor of the present invention and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of the bile acid ttansport inhibitor of the present invention. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The bile acid transport inhibitor of the present invention is the subject of WO 96/5188 (1996), based on US application 288527 (1994), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises the bile acid ttansport inhibitor of the present invention covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturaUy occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and CW080P

rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial arrangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stabihty is the soUd reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stabiUty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is CW080P

a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of i eversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Upophilicity is desired, then the carrier polypeptide wUl be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired. CW080P

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. GeneraUy, it was only possible to load up to 1/10 of the total drug-dexfran conjugate weight with drug. As stated earUer, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW080P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

1 1 CW080P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

12 CW080P

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copoljrmerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions,

13 CW080P

sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bUe acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-bile acid transport inhibitor conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours

14 CW080P

at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, uiethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl gfutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for

15 CW080P

several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the detaUs shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW080P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and a bUe acid transport inhibitor covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an ohgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein the bile acid ttansport inhibitor is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW080P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein the bile acid fransport inhibitor is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing the bile acid transport inhibitor from said composition in a pH-dependent manner.

19. A method for protecting a bile acid ttansport inhibitor from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of a bile acid transport inhibitor from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching the bile acid ttansport inhibitor to said polypeptide.

21. A method for delivering a bile acid ttansport inhibitor to a patient comprising administering to said patient a composition comprising: a polypeptide; and a bile acid transport inhibitor covalently attached to said polypeptide.

22. The method of claim 21 wherein the bile acid transport inhibitor is released from said composition by an enzyme-catalyzed release.

18 CW080P

23. The method of claim 21 wherein the bile acid ttansport inhibitor is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and a bUe acid fransport inhibitor covalently attached to the polypeptide. Also provided is a method for delivery of a bile acid transport inhibitor to a patient comprising administering to the patient a composition comprising a polypeptide and a bile acid transport inhibitor covalently attached to the polypeptide. Also provided is a method for protecting a bile acid transport inhibitor from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of a bile acid ttansport inhibitor from a composition comprising covalentiy attaching it to the polypeptide.

19 CW081P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFUROXIME AXETILAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to cefuroxime axetil, as well as methods for protecting and administering cefuroxime axetil. This novel compound, referred to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefuroxime axetil is a known pharmaceutical agent that is used in the treatment of bacterial infection. Its chemical name is [6R-[6alpha,7beta(Z)]]-3-[[(aminocarbonyl) oxy]methyl]-7-[[2-furan yl (methoxyimino)acetyl]amino]-8-oxo-5-thia-l- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, l-(acetyloxy)ethyl ester. Its structure is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW081P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stabihty of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabiUty of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW081P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW081P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cefuroxime axetil) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cefuroxime axetil to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, deUvery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefuroxime axetil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefuroxime axetil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cefuroxime axetil preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active CW081P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalentiy attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefuroxime axetil from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cefuroxime axetil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a poljφeptide and an active agent covalentiy attached to the polypeptide. In a prefeoed embodiment, cefuroxime axetil is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, cefuroxime axetil is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cefuroxime axetil is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cefuroxime axetil is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cefuroxime axetil is CW081P

released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching cefuroxime axetil to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefuroxime axetil and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is CW081P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cefuroxime axetil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefuroxime axetil. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cefuroxime axetil is the subject of GB 1571683 (1980), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cefuroxime axetil covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the poljφeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW081P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystaUine solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stabihty of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW081P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophihcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but wUl ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given apphcation. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the poljφeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW081P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. GeneraUy, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controUed and, thus, active agent loading can also be controlled.

As a practical example, the foUowing table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW081P

delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-teiminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teiminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW081P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or tinker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-teπninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cefuroxime axetil is covalently attached to the polypeptide via the amido group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

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The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in locaUzed delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefuroxime axetil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

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Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with dusopropylcarbodUmide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW081P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW081P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and cefuroxime axetil covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefuroxime axetil is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW081P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefuroxime axetil is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefuroxime axetil from said composition in a pH-dependent manner.

19. A method for protecting cefuroxime axetil from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cefuroxime axetil from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cefuroxime axetil to said polypeptide.

21. A method for delivering cefuroxime axetil to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefuroxime axetil covalently attached to said polypeptide.

22. The method of claim 21 wherein cefuroxime axetil is released from said composition by an enzyme-catalyzed release.

17 CW081P

23. The method of claim 21 wherein cefuroxime axetil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW081P

Abstract

A composition comprising a polypeptide and cefuroxime axetil covalently attached to the polypeptide. Also provided is a method for delivery of cefuroxime axetil to a patient comprising administering to the patient a composition comprising a polypeptide and cefuroxime axetil covalently attached to the polypeptide. Also provided is a method for protecting cefuroxime axetil from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cefuroxime axetil from a composition comprising covalently attaching it to the polypeptide.

19 CW082P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEFUROXIMEAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalentiy attached to cefuroxime, as well as methods for protecting and administering cefuroxime. This novel compound, refened to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cefuroxime is a known pharmaceutical agent that is used in the treatment of bacterial infection. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW082P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabUity of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW082P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspaitic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW082P

Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(cefuroxime) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cefuroxime to the N-terminus, the C-terminus or directly to the amino acid side chain of an oUgopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabUize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cefuroxime microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cefuroxime covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Cefuroxime preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW082P

the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cefuroxime from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering cefuroxime to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefeoed embodiment, cefuroxime is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cefuroxime is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and cefuroxime is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, cefuroxime is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, cefuroxime is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant CW082P

from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching cefuroxime to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cefuroxime and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW082P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cefuroxime and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cefuroxime. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cefuroxime covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturaUy occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial anangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stabihty is the solid reference state. CW082P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the canier polypeptide will be CW082P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proUne, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the canier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular canier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW082P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW082P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-teoninus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular ttansamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

11 CW082P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the poljφeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not hmited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide Unked acridine.

In the present invention, cefuroxime is covalentiy attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW082P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cefuroxime conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW082P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with dusopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination Of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-teoninus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW082P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW082P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cefuroxime covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cefuroxime is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW082P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cefuroxime is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cefuroxime from said composition in a pH-dependent manner.

19. A method for protecting cefuroxime from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cefuroxime from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cefuroxime to said polypeptide.

21. A method for delivering cefuroxime to a patient comprising administering to said patient a composition comprising: a polypeptide; and cefuroxime covalently attached to said polypeptide.

22. The method of claim 21 wherein cefuroxime is released from said composition by an enzyme-catalyzed release.

17 CW082P

23. The method of claim 21 wherein cefuroxime is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW082P

Abstract

A composition comprising a polypeptide and cefuroxime covalently attached to the polypeptide. Also provided is a method for delivery of cefuroxime to a patient comprising administering to the patient a composition comprising a polypeptide and cefuroxime covalently attached to the polypeptide. Also provided is a method for protecting cefuroxime from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cefuroxime from a composition comprising covalently attaching it to the polypeptide.

19 CW083P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CELECOXffiAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to celecoxib, as well as methods for protecting and administering celecoxib. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Celecoxib is a known pharmaceutical agent that is used in the treatment of osteo- and rheumatoid arthritis. Its chemical name is 4-[5-(4-methylphenyl)-3- (trifluoromethyl)-lH-pyrazol-l-yl]benzenesulfonamide. Its stracmre is:

o — s — 0

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical CW083P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, wiU assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW083P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent avaUable for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an entericaUy coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW083P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR apphcation, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (celecoxib) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching celecoxib to the N- teoninus, the C-teoninus or directly to the amino acid side chain of an oUgopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising celecoxib microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and celecoxib covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW083P

Celecoxib preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationaUy protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting celecoxib from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering celecoxib to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, celecoxib is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, celecoxib is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and celecoxib is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, celecoxib is released CW083P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, celecoxib is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of: (a) attaching celecoxib to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, celecoxib and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutaπuc acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW083P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize celecoxib and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of celecoxib. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Celecoxib is the subject of U.S. Patent Numbers 5,466,823, 5,563,165, 5,760,068 and 5,972,986, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises celecoxib covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oUgopeptide, (ii) a homopolymer of one of the twenty naturaUy occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW083P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are estabUshed during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW083P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH confroUed peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but wiU ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW083P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug deUvery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of Upophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW083P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oUgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oUgopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

11 CW083P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the poljφeptide using known techniques. Examples of Unking organic compounds to the N-terminus type of a peptide include, but are not hmited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalentiy attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW083P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabUizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized ttansporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-celecoxib conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW083P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodumide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW083P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, ttiethylamine or ttibutylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW083P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW083P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and celecoxib covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oUgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occuning amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein celecoxib is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW083P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein celecoxib is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing celecoxib from said composition in a pH-dependent manner.

19. A method for protecting celecoxib from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of celecoxib from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching celecoxib to said polypeptide.

21. A method for delivering celecoxib to a patient comprising administering to said patient a composition comprising: a polypeptide; and celecoxib covalently attached to said polypeptide.

22. The method of claim 21 wherein celecoxib is released from said composition by an enzyme-catalyzed release. CW083P

23. The method of claim 21 wherein celecoxib is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and celecoxib covalently attached to the polypeptide. Also provided is a method for delivery of celecoxib to a patient comprising administering to the patient a composition comprising a polypeptide and celecoxib covalently attached to the polypeptide. Also provided is a method for protecting celecoxib from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of celecoxib from a composition comprising covalently attaching it to the polypeptide.

19 CW084P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CEPHALEXINAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cephalexin, as well as methods for protecting and administering cephalexin. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cephalexin is a known pharmaceutical agent that is used in the treatment of bacterial infection. It is both commercially available and readUy manufactured using published synthetic schemes by those of ordinary skiU in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW084P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stabiUty of the active agent, such as prolonging shelf life or survival in the stomach, wiU assure dosage reproducibiUty and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bUe acids enhance permeability of ceUular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW084P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug deUvery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW084P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cephalexin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching cephalexin to the N-terminus, the C-terminus or directly to the amino acid side chain of an ohgopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain appUcations, the polypeptide will stabiUze the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cephalexin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cephalexin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cephalexin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW084P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationaUy protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cephalexin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cephalexin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefened embodiment, cephalexin is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cephalexin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cephalexin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cephalexin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cephalexin is released from the composition in a sustained CW084P

release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching cephalexin to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cephalexin and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW084P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cephalexin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cephalexin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cephalexin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW084P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stabiUty of the folded protein where the degree to which ideal packing occurs determines the degree of relative stabiUty of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW084P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but wiU ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crossUnking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW084P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW084P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW084P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the poljφeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cephalexin is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW084P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bUe acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cephalexin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW084P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The cmde product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopjridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW084P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alk l Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW084P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cephalexin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cephalexin is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW084P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cephalexin is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cephalexin from said composition in a pH-dependent manner.

19. A method for protecting cephalexin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cephalexin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cephalexin to said polypeptide.

21. A method for delivering cephalexin to a patient comprising administering to said patient a composition comprising: a polypeptide; and cephalexin covalently attached to said polypeptide.

22. The method of claim 21 wherein cephalexin is released from said composition by an enzyme-catalyzed release.

17 CW084P

23. The method of claim 21 wherein cephalexin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW084P

Abstract

A composition comprising a polypeptide and cephalexin covalentiy attached to the polypeptide. Also provided is a method for delivery of cephalexin to a patient comprising administering to the patient a composition comprising a polypeptide and cephalexin covalentiy attached to the polypeptide. Also provided is a method for protecting cephalexin from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of cephalexin from a composition comprising covalently attaching it to the polypeptide.

19 CW085P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CERIVASTATINAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to cerivastatin, as well as methods for protecting and administering cerivastatin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both weU smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cerivastatin is a known pharmaceutical agent that is used in the tteatment of cholesterolemia. Its chemical name is [S-[R*,S*-(E)]]-7-[4-(4-fluorophenyl)-5-

(methoxymethyl)-2,6-bis ( 1 -methylethyl)-3-pyridinyl]-3,5-dihydroxy-6-heptenoic acid. Its stracmre is:

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW085P

compound may contain one or more of the following: another active phaonaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and ttansport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW085P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typicaUy require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW085P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is Umited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cerivastatin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cerivastatin to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide wUl stabUize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cerivastatin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cerivastatin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW085P

Cerivastatin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-teιτninus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the C-teoninus of the poljφeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cerivastatin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cerivastatin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, cerivastatin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cerivastatin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cerivastatin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cerivastatin is CW085P

released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cerivastatin is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cerivastatin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cerivastatin and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW085P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cerivastatin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cerivastatin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cerivastatin is the subject of U.S. Patent Number 5,177,080, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises cerivastatin covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture. CW085P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stabihty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Tjφically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW085P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stabiUty of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Upophilicity is desired, then the carrier poljφeptide wiU be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW085P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controUed.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an ohgopeptide. Eighteen was subtracted from the amino acid's

10 CW085P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oUgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terniinus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active

11 CW085P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cerivastatin is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW085P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophitic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized deUvery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cerivastatin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW085P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AcidVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with dusopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW085P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AlkyI Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW085P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW085P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cerivastatin covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cerivastatin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW085P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cerivastatin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cerivastatin from said composition in a pH-dependent manner.

19. A method for protecting cerivastatin from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of cerivastatin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cerivastatin to said polypeptide.

21. A method for delivering cerivastatin to a patient comprising administering to said patient a composition comprising: a polypeptide; and cerivastatin covalently attached to said polypeptide.

22. The method of claim 21 wherein cerivastatin is released from said composition by an enzyme-catalyzed release.

18 CW085P

23. The method of claim 21 wherein cerivastatin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and cerivastatin covalently attached to the polypeptide. Also provided is a method for delivery of cerivastatin to a patient comprising administering to the patient a composition comprising a poljφeptide and cerivastatin covalentiy attached to the polypeptide. Also provided is a method for protecting cerivastatin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cerivastatin from a composition comprising covalently attaching it to the polypeptide.

19 CW086P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CETIRIZINEAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cetirizine, as weU as methods for protecting and administering cetirizine. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a canier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cetirizine is a known pharmaceutical agent that is used in the treatment of allergic rhinitis. Its chemical name is [2-[4-[(4-chlorophenyl)phenylmethyl]-l-piperazinyl] ethoxy]acetic acid. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW086P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabiUty are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeabiUty of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW086P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW086P

the colon by bacterial enzymes. High molecular weight microspheres usuaUy have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cetirizine) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalentiy attaching cetirizine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cetirizine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cetirizine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cetirizine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW086P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cetirizine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cetirizine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cetirizine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cetirizine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and cetirizine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, cetirizine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, cetirizine is released from the composition in a sustained release. CW086P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cetirizine to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cetirizine and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW086P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize cetirizine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cetirizine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cetirizine is the subject of U.S. Patent Number 4,525,358, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cetirizine covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constimte the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW086P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW086P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophtiicity of the poljφeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW086P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW086P

delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teπriinus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicUUn), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide canier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW086P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not hmited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the poljφeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not Umited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cetirizine is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW086P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane fransport system is intrinsicaUy asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of speciaUzed adjuvant resulting in localized deUvery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the ttansported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cetirizine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW086P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW086P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW086P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and cetirizine covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cetirizine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW086P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cetirizine is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cetirizine from said composition in a pH-dependent manner.

19. A method for protecting cetirizine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cetirizine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cetirizine to said polypeptide.

21. A method for delivering cetirizine to a patient comprising administering to said patient a composition comprising: a polypeptide; and cetirizine covalently attached to said polypeptide.

22. The method of claim 21 wherein cetirizine is released from said composition by an enzyme-catalyzed release.

17 CW086P

23. The method of claim 21 wherein cetirizine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW086P

Abstract

A composition comprising a polypeptide and cetirizine covalently attached to the poljφeptide. Also provided is a method for delivery of cetirizine to a patient comprising administering to the patient a composition comprising a polypeptide and cetirizine covalently attached to the polypeptide. Also provided is a method for protecting cetirizine from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for conttolling release of cetirizine from a composition comprising covalentiy attaching it to the polypeptide.

19 CW087P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CHLORAZEPATE DEPOTAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to chlorazepate depot, as well as methods for protecting and administering chlorazepate depot. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Chlorazepate depot is a known pharmaceutical agent that is used in the treatment of anxiety disorders. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW087P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW087P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW087P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brash-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (chlorazepate depot) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching chlorazepate depot to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or poljφeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is confroUed, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising chlorazepate depot microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and chlorazepate depot covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids. CW087P

Chlorazepate depot preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationaUy protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting chlorazepate depot from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering chlorazepate depot to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, chlorazepate depot is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, chlorazepate depot is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and chlorazepate depot is released from the composition by dissolution of the microencapsulating agent. In another prefened CW087P

embodiment, chlorazepate depot is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, chlorazepate depot is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching chlorazepate depot to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, chlorazepate depot and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutaπuc acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW087P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize chlorazepate depot and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of chlorazepate depot. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises chlorazepate depot covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oUgopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constimte the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW087P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are estabUshed during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW087P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but wiU ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW087P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the Ueum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW087P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teiminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig.4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular ttansamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW087P

any suitable initiator as shown in Fig.4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polyt Tosine, poljrthreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teπninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, chlorazepate depot is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide canier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW087P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the ttansported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-chlorazepate depot conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW087P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW087P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW087P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and chlorazepate depot covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said poljφeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturaUy occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said poljφeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein chlorazepate depot is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW087P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein chlorazepate depot is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing chlorazepate depot from said composition in a pH-dependent manner.

19. A method for protecting chlorazepate depot from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of chlorazepate depot from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching chlorazepate depot to said polypeptide.

21. A method for delivering chlorazepate depot to a patient comprising administering to said patient a composition comprising: a polypeptide; and chlorazepate depot covalently attached to said polypeptide.

22. The method of claim 21 wherein chlorazepate depot is released from said composition by an enzyme-catalyzed release.

17 CW087P

23. The method of claim 21 wherein chlorazepate depot is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW087P

Abstract

A composition comprising a polypeptide and chlorazepate depot covalentiy attached to the polypeptide. Also provided is a method for delivery of chlorazepate depot to a patient comprising administering to the patient a composition comprising a polypeptide and chlorazepate depot covalently attached to the polypeptide. Also provided is a method for protecting chlorazepate depot from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controUing release of chlorazepate depot from a composition comprising covalentiy attaching it to the polypeptide.

19 CW088P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CHLORDIAZEPOXIDEAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to chlordiazepoxide, as well as methods for protecting and administering chlordiazepoxide. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Chlordiazepoxide is a known pharmaceutical agent that is used in the treatment of anxiety and tension. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; CW088P

and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations. CW088P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW088P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usuaUy have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (chlordiazepoxide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching chlordiazepoxide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or poljφeptide, also referred to herein as a carrier peptide. In certain applications, the poljφeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising chlordiazepoxide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and chlordiazepoxide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic CW088P

amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Chlordiazepoxide preferably is covalentiy attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- teiminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting chlordiazepoxide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering chlordiazepoxide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, chlordiazepoxide is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, chlordiazepoxide is released in a time-dependent manner based on the pharmacokinetics CW088P

of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and chlordiazepoxide is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, chlordiazepoxide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, chlordiazepoxide is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controUed by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching chlordiazepoxide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, chlordiazepoxide and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the CW088P

glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detaUed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize chlordiazepoxide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of chlordiazepoxide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises chlordiazepoxide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oUgopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture. CW088P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW088P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW088P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controUed.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW088P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the ohgopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW088P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig.4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these poljφeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-teπninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, chlordiazepoxide is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW088P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. StabUizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-chlordiazepoxide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW088P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW088P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutaπuc acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystalUzed from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alky 1 Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW088P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW088P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and chlordiazepoxide covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturaUy occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein chlordiazepoxide is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW088P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein chlordiazepoxide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing chlordiazepoxide from said composition in a pH-dependent manner.

19. A method for protecting chlordiazepoxide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of chlordiazepoxide from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching chlordiazepoxide to said polypeptide.

21. A method for delivering chlordiazepoxide to a patient comprising administering to said patient a composition comprising: a polypeptide; and chlordiazepoxide covalently attached to said polypeptide.

22. The method of claim 21 wherein chlordiazepoxide is released from said composition by an enzyme-catalyzed release.

18 CW088P

23. The method of claim 21 wherein chlordiazepoxide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and chlordiazepoxide covalently attached to the polypeptide. Also provided is a method for delivery of chlordiazepoxide to a patient comprising administering to the patient a composition comprising a poljφeptide and chlordiazepoxide covalentiy attached to the polypeptide. Also provided is a method for protecting chlordiazepoxide from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for conttolling release of chlordiazepoxide from a composition comprising covalently attaching it to the polypeptide.

19 CW089P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CICLESONIDEAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to ciclesonide, as well as methods for protecting and administering ciclesonide. This novel compound, refeoed to as a CARRLERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ciclesonide is a known phaonaceutical agent that is used in the treatment of asthma. Its chemical name is [1 lbeta,16alfa (R)]-16,17-

[(cyclohexylmethylene)bis(oxy)]- 11 -hydroxy-21 -(2-methyl- 1 -oxopropoxy)-pregna- 1 ,4- diene-3,20-dione. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW089P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compUance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf Ufe or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and ttansport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid πucrospheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW089P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with Uttle active agent avaUable for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typicaUy require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW089P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ciclesonide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ciclesonide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oUgopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ciclesonide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ciclesonide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ciclesonide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW089P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationaUy protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ciclesonide from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering ciclesonide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, ciclesonide is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, ciclesonide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ciclesonide is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, ciclesonide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ciclesonide is released from the composition in a CW089P

sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ciclesonide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ciclesonide and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular ttansamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW089P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ciclesonide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ciclesonide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Ciclesonide is the subject of GB 2247680 B (1994), based on US Apphcation

Number 578942 (1990), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises ciclesonide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and pol φeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW089P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW089P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crossUnking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW089P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW089P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or poljφeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicUlin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW089P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, rrølythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the poljφeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ciclesonide is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW089P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via speciaUzed ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ciclesonide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW089P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW089P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW089P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ciclesonide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said poljφeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said poljφeptide is a heteropolymer of one or more namrally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein ciclesonide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW089P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ciclesonide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said poljφeptide is capable of releasing ciclesonide from said composition in a pH-dependent manner.

19. A method for protecting ciclesonide from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of ciclesonide from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching ciclesonide to said polypeptide.

21. A method for delivering ciclesonide to a patient comprising administering to said patient a composition comprising: a polypeptide; and ciclesonide covalently attached to said polypeptide.

22. The method of claim 21 wherein ciclesonide is released from said composition by an enzyme-catalyzed release.

17 CW089P

23. The method of claim 21 wherein ciclesonide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW089P

Abstract

A composition comprising a polypeptide and ciclesonide covalently attached to the polypeptide. Also provided is a method for delivery of ciclesonide to a patient comprising administering to the patient a composition comprising a polypeptide and ciclesonide covalently attached to the polypeptide. Also provided is a method for protecting ciclesonide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controUing release of ciclesonide from a composition comprising covalently attaching it to the polypeptide.

19 CW090P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CILANSETRONAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cilansetton, as well as methods for protecting and administering ctiansetron. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cilansetton is a known pharmaceutical agent that is used in the treatment of irritable bowel syndrome. Its chemical name is (R)-5,6,9,10-tettahydro-10-[(2-methyl- lH-imidazol-l-yl)methyl]-4H-pyrido[3,2,l-jk]carbazol-l l(8H)-one. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW090P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bUe acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, Uposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent deUvery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW090P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with Uttle active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW090P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cUansetton) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cilansetton to the N-terminus, the C-teoninus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these apphcations, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cilansetton microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cilansefron covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Cilansetton preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW090P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cilansetton from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering cilansetton to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, cilansetton is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cilansetton is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cilansetton is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cilansetton is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cilansetton is released from the composition in a CW090P

sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cilansetron to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cilansetron and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular ttansamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW090P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cilansetron and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cilansetron. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also aUows targeted delivery of active agents to specifics sites of action.

Cilansetron is the subject of EP 297651 (1989), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cUansetron covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW090P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline soUd than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stabiUty of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW090P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controUed peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosshnking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW090P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW090P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

^' poly(hydroxjφropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide canier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW090P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide canier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW090P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epitheha efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cilansetron conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW090P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with ctiisopropylcarbodurnide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or ttibutylamine.

14 CW090P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodumide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW090P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and cilansetron covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cilansetron is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW090P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cUansetton is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cilansetton from said composition in a pH-dependent manner.

19. A method for protecting cilansetron from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of cilansetton from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching cilansetton to said polypeptide.

21. A method for delivering cilansetron to a patient comprising administering to said patient a composition comprising: a polypeptide; and cilansetron covalently attached to said polypeptide.

22. The method of claim 21 wherein cilansetron is released from said composition by an enzyme-catalyzed release.

17 CW090P

23. The method of claim 21 wherein cilansetton is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW090P

Abstract

A composition comprising a polypeptide and cilansetton covalentiy attached to the polypeptide. Also provided is a method for delivery of cilansetton to a patient comprising administering to the patient a composition comprising a polypeptide and cilansetron covalentiy attached to the polypeptide. Also provided is a method for protecting cilansetton from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cilansetton from a composition comprising covalentiy attaching it to the polypeptide.

19 CW091P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING

CILASTATIN AND EVflPENEMAND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to cilastatin and imipenem, as well as methods for protecting and administering cilastatin and imipenem. This novel compound, refeoed to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cilastatin and imipenem are known phaonaceutical agents that are used together in the treatment of bacterial infections. Cilastatin has no antibacterial activity, but increases the effectiveness of imipenem. Each is commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. The stracture of imipenem is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW091P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent deUvery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered oraUy in Ueu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, wUl assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compUance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW091P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreUable. For example, an entericaUy coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW091P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cilastatin and imipenem) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cilastatin and imipenem to the N-teoninus, the C-teoninus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain appUcations, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controUed, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising cilastatin and imipenem microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cilastatin and imipenem covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW091P

Cilastatin and imipenem preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalentiy attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the poljφeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cilastatin and imipenem from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cilastatin and imipenem to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, cilastatin and imipenem are released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cilastatin and imipenem are released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cilastatin and imipenem are released from the composition by dissolution of the microencapsulating CW091P

agent. In another prefeoed embodiment, cilastatin and imipenem are released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, cilastatin and imipenem are released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching cilastatin and imipenem to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cilastatin and imipenem and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW091P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cilastatin and imipenem and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cilastatin and imipenem. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cilastatin and imipenem covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oUgopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW091P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW091P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW091P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the Ueum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW091P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide canier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutaπuc acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N-

11 CW091P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is teπmnated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety rehes on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, imipenem is covalently attached to the polypeptide via the carboxylic acid. Ciliastatin can be attached via any free alcohol, acid, or amine group, or can be attached via a linker.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW091P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the ttansported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also aUows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cilastatin and imipenem conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW091P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodumide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW091P

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW091P

What is claimed is:

1. A pharmaceutical composition comprising: a poljφeptide; and cilastatin and imipenem covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more sjmthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cilastatin and imipenem are covalently attached to a side chain, the N-terminus or the C-teoninus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW091P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cilastatin and imipenem are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cilastatin and imipenem from said composition in a pH-dependent manner.

19. A method for protecting cilastatin and imipenem from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cilastatin and imipenem from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cilastatin and imipenem to said polypeptide.

21. A method for delivering cilastatin and imipenem to a patient comprising administering to said patient a composition comprising: a polypeptide; and cilastatin and imipenem covalently attached to said polypeptide.

22. The method of claim 21 wherein cilastatin and imipenem are released from said composition by an enzyme-catalyzed release.

17 CW091P

23. The method of claim 21 wherein cilastatin and imipenem are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW091P

Abstract

A composition comprising a polypeptide and cilastatin and imipenem covalentiy attached to the polypeptide. Also provided is a method for delivery of cUastatin and imipenem to a patient comprising administering to the patient a composition comprising a polypeptide and cilastatin and imipenem covalently attached to the polypeptide. Also provided is a method for protecting cilastatin and imipenem from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for conttolling release of cilastatin and imipenem from a composition comprising covalently attaching it to the polypeptide.

19 CW092P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CILOMILASTAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cilomUast, as weU as methods for protecting and administering cilomUast. This novel compound, referred to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

CilomUast is a known pharmaceutical agent that is used in the treatment of asthma. Its chemical name is cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl] cyclohexanecarboxylic acid. Its stracture is:

The novel pha naceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW092P

Active agent delivery systems are often critical for the effective delivery of a biologicaUy active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compUance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bUe acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW092P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug deUvery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW092P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cilomUast) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cilomUast to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cilomUast microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cilomUast covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

CilomUast preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW092P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-teπriinus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cilomUast from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cilomUast to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cilomUast is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cilomUast is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and cilomUast is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, cilomUast is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, cilomUast is released from the composition in a sustained release. CW092P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cilomilast to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cilomilast and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is CW092P

described in U.S. Patent Apphcation Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cilomilast and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cilomilast. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cilomilast is the subject of WO 93/19749 (1993), based on priority US application 862030 (1992), herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises cilomilast covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW092P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oU drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW092P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier poljφeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophiUcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW092P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. GeneraUy, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an ohgopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW092P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW092P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-teoninus, the C-teoninus or the side chain of the poljφeptide using known techniques. Examples of Unking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cilomilast is covalentiy attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW092P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cilomilast conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW092P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW092P

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the rnixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW092P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cilomilast covalently attached to said polypeptide.

2. The composition of claim 1 wherein said poljφeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein cilomilast is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW092P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cilomilast is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cilomilast from said composition in a pH-dependent manner.

19. A method for protecting cilomilast from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cilomilast from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cilomilast to said polypeptide.

21. A method for delivering cilomilast to a patient comprising administering to said patient a composition comprising: a polypeptide; and cilomilast covalently attached to said polypeptide.

22. The method of claim 21 wherein cilomilast is released from said composition by an enzyme-catalyzed release.

17 CW092P

23. The method of claim 21 wherein cilomilast is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW092P

Abstract

A composition comprising a polypeptide and cilomilast covalently attached to the polypeptide. Also provided is a method for delivery of cilomilast to a patient comprising administering to the patient a composition comprising a polypeptide and cilomUast covalentiy attached to the polypeptide. Also provided is a method for protecting cilomilast from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cilomilast from a composition comprising covalentiy attaching it to the polypeptide.

19 CW093P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CIMETIDINEAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to cimetidine, as weU as methods for protecting and administering cimetidine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well smdied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cimetidine is a known pharmaceutical agent that is used in the treatment of duodenal ulcer. It is both commercially available and readily manufactured using pubUshed sjmthetic schemes by those of ordinary skill in the art. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW093P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, hposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW093P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW093P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cimetidine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching cimetidine to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzj natic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cimetidine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cimetidine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cimetidine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW093P

carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cimetidine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cimetidine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cimetidine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cimetidine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and cimetidine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, cimetidine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cimetidine is released from the composition in a sustained release. CW093P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching cimetidine to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cimetidine and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is CW093P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cimetidine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cimetidine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cimetidine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (Ui) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW093P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the sohd reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are estabUshed during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stabihty of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophUic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW093P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can aU be used to select the optimum amino acid sequence for a given apphcation. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW093P

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be conttoUed and, thus, active agent loading can also be controlled.

As a practical example, the following table Usts the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW093P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teraiinus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW093P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cimetidine is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW093P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epitheha efficientiy via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oUgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cimetidine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW093P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW093P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the inixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW093P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and cimetidine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cimetidine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW093P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cimetidine is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cimetidine from said composition in a pH-dependent manner.

19. A method for protecting cimetidine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of cimetidine from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching cimetidine to said polypeptide.

21. A method for delivering cimetidine to a patient comprising administering to said patient a composition comprising: a polypeptide; and cimetidine covalently attached to said polypeptide.

22. The method of claim 21 wherein cimetidine is released from said composition by an enzyme-catalyzed release.

17 CW093P

23. The method of claim 21 wherein cimetidine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW093P

Abstract

A composition comprising a polypeptide and cimetidine covalently attached to the poljφeptide. Also provided is a method for delivery of cimetidine to a patient comprising administering to the patient a composition comprising a polypeptide and cimetidine covalentiy attached to the polypeptide. Also provided is a method for protecting cimetidine from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for conttolling release of cimetidine from a composition comprising covalently attaching it to the polypeptide.

19 CW094P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CIPROFLOXACINAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ciprofloxacin, as well as methods for protecting and administering ciprofloxacin. This novel compound, referred to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ciprofloxacin is a known pharmaceutical agent that is used in the treatment of bacterial infection. Its chemical name is l-cyclopropyl-6-fluoro-l,4-dihydro-4-oxo-7-(l- piperazinyl)-3-quinolinecarboxylic acid. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor. CW094P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copol mier of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW094P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in CW094P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR apphcation, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ciprofloxacin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ciprofloxacin to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabUize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzjmies release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising ciprofloxacin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ciprofloxacin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ciprofloxacin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the poljφeptide. In a prefened embodiment, the active agent is a CW094P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationaUy protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ciprofloxacin from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering ciprofloxacin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, ciprofloxacin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, ciprofloxacin is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ciprofloxacin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, ciprofloxacin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ciprofloxacin is released from the CW094P

composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ciprofloxacin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent amino acid complex N-cartκ xyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ciprofloxacin and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a sjmthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW094P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ciprofloxacin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ciprofloxacin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Ciprofloxacin is the subject of U.S. Patent Numbers 4,670,444 and 5,286,754, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises ciprofloxacin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopoljmier of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW094P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stabihty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the soUd reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW094P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophihcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW094P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug deUvery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW094P

delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oUgopeptide or poljφeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW094P

any suitable initiator as shown in Fig.4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-teiminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ciprofloxacin is covalently attached to the polypeptide via the carboxylic acid.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW094P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized fransporters. The entire membrane fransport system is intrinsicaUy asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, ohgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ciprofloxacin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW094P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaπύnopyridine, triethylamine or tributylamine.

14 CW094P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW094P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and ciprofloxacin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturaUy occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ciprofloxacin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW094P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceuticaUy acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ciprofloxacin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ciprofloxacin from said composition in a pH-dependent manner.

19. A method for protecting ciprofloxacin from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of ciprofloxacin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ciprofloxacin to said polypeptide.

21. A method for delivering ciprofloxacin to a patient comprising administering to said patient a composition comprising: a polypeptide; and ciprofloxacin covalently attached to said polypeptide.

22. The method of claim 21 wherein ciprofloxacin is released from said composition by an enzyme-catalyzed release.

17 CW094P

23. The method of claim 21 wherein ciprofloxacin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW094P

Abstract

A composition comprising a polypeptide and ciprofloxacin covalently attached to the polypeptide. Also provided is a method for delivery of ciprofloxacin to a patient comprising administering to the patient a composition comprising a polypeptide and ciprofloxacin covalently attached to the polypeptide. Also provided is a method for protecting ciprofloxacin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ciprofloxacin from a composition comprising covalentiy attaching it to the polypeptide.

19 CW095P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CISAPRIDEAND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cisapride, as well as methods for protecting and administering cisapride. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cisapride is a known pharmaceutical agent that is used in the treatment of gastrointestinal motility disease. Its chemical name is cis-4-amino-5-chloro-N-[l-[3-(4- fluorophenoxy)propyl]-3-methoxy-4-piperidinyl]-2-methoxybenzamide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW095P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabUity of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW095P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag deUvery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW095P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cisapride) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching cisapride to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cisapride microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cisapride covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cisapride preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW095P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cisapride from degradation comprising covalentiy attaching it to a poljφeptide.

The invention also provides a method for delivering cisapride to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cisapride is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cisapride is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and cisapride is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, cisapride is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, cisapride is released from the composition in a sustained release. CW095P

In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cisapride to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cisapride and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW095P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cisapride and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cisapride. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cisapride is the subject of U.S. Patent Number 4,962,115, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cisapride covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iU) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW095P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an₍oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW095P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW095P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly Umited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW095P

delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teπninus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teπninus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW095P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not hmited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the poljφeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cisapride is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW095P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system wiU involve some sort of specialized adjuvant resulting in localized deUvery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oUgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cisapride conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW095P

AcidVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

AIcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylaπύne.

14 CW095P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW095P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cisapride covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cisapride is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW095P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cisapride is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cisapride from said composition in a pH-dependent manner.

19. A method for protecting cisapride from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cisapride from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching cisapride to said polypeptide.

21. A method for delivering cisapride to a patient comprising administering to said patient a composition comprising: a polypeptide; and cisapride covalently attached to said polypeptide.

22. The method of claim 21 wherein cisapride is released from said composition by an enzyme-catalyzed release.

17 CW095P

23. The method of claim 21 wherein cisapride is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW095P

Abstract

A composition comprising a polypeptide and cisapride covalentiy attached to the polypeptide. Also provided is a method for delivery of cisapride to a patient comprising administering to the patient a composition comprising a poljφeptide and cisapride covalentiy attached to the polypeptide. Also provided is a method for protecting cisapride from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for conttolling release of cisapride from a composition comprising covalentiy attaching it to the polypeptide.

19 CW096P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING

CISATRACURIUM BESYLATEAND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to cisatracurium besylate, as weU as methods for protecting and administering cisatracurium besylate. This novel compound, referred to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both weU studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cisatracurium besylate is a known pharmaceutical agent that is used as a neuromuscular blocker in surgery. Its chemical name is [lR-[lalpha,2alpha(lR*,2'R*)]] -2,2^,-[l,5-p^ntanediylbis[oxy(3-oxoo-3,l-propanediyl)]]bis[l-[(3,4-dimethoxyphenyl) methyl]-!, 2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-isoquinolinium. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stabihty of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; CW096P

and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabUity are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stabiUty of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibiUty and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and fransport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations. CW096P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreUable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: Unking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW096P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cisatracurium besylate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cisatracurium besylate to the N-terminus, the C-terminus or directly to the amino acid side chain of an ohgopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising cisatracurium besylate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cisatracurium besylate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or CW096P

(vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Cisatracurium besylate preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the poljφeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cisatracurium besylate from degradation comprising covalently attaching it to a poljφeptide.

The invention also provides a method for delivering cisatracurium besylate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefeoed embodiment, cisatracurium besylate is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cisatracurium besylate is released in a time-dependent manner based on the CW096P

pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cisatracurium besylate is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cisatracurium besylate is released from the composition by a pH- dependent unfolding of the polypeptide. In another preferred embodiment, cisatracurium besylate is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cisatracurium besylate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) poljm erizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cisatracurium besylate and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the CW096P

glutamic acid is replaced by a sjmthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cisatracurium besylate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cisatracurium besylate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cisatracurium besylate is the subject of U.S. Patent Number 5,453,510 and WO 92/965 (1992), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cisatracurium besylate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oUgopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The CW096P

folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stabUity, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and CW096P

at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophiUcity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain CW096P

length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug deUvery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earUer, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the Ueum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW096P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

11 CW096P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue wtil then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This canier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW096P

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide Unked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions,

13 CW096P

sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bUe acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cisattacurium besylate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

14 CW096P

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiiinide or thionyl chloride. An example of another cocatalyst is N- hydroxysuecinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or ttibutylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW096P

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typicaUy overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW096P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cisatracurium besylate covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more sjmthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cisatracurium besylate is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW096P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cisatracurium besylate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cisatracurium besylate from said composition in a pH-dependent manner.

19. A method for protecting cisatracurium besylate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of cisatracurium besylate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cisatracurium besylate to said polypeptide.

21. A method for delivering cisatracurium besylate to a patient comprising administering to said patient a composition comprising: a polypeptide; and cisatracurium besylate covalently attached to said polypeptide.

22. The method of claim 21 wherein cisatracurium besylate is released from said composition by an enzyme-catalyzed release.

18 CW096P

23. The method of claim 21 wherein cisatracurium besylate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and cisatracurium besylate covalently attached to the polypeptide. Also provided is a method for delivery of cisatracurium besylate to a patient comprising administering to the patient a composition comprising a polypeptide and cisatracurium besylate covalentiy attached to the polypeptide. Also provided is a method for protecting cisattacurium besylate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cisattacurium besylate from a composition comprising covalentiy attaching it to the polypeptide.

19 CW097P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CISPLATINAND METHODS OF MAKING AND USING SAME

FTELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cisplatin, as well as methods for protecting and administering cisplatin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cisplatin is a known phaimaceutical agent that is used in the treatment of bladder and ovarian carcinoma. It is both commercially available and readily manufactured using published sjmthetic schemes by those of ordinary skill in the art. Its stracmre is:

Pt

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; CW097P

and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabiUty are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf hfe or survival in the stomach, will assure dosage reproducibiUty and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabiUty of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolj ier of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations. CW097P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with tittle active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW097P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is hmited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cisplatin) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalentiy attaching cisplatin to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide wUl stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzjmies release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cisplatin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cisplatin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW097P

heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cisplatin preferably is covalentiy attached to a side chain, the N-terminus or the C-teoninus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cisplatin from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering cisplatin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cisplatin is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cisplatin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. CW097P

In another prefened embodiment, the composition further comprises a microencapsulating agent and cisplatin is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, cisplatin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, cisplatin is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cisplatin to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cisplatin and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW097P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cisplatin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cisplatin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cisplatin is the subject of U.S. Patent Number 5,562,925, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises cisplatin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture. CW097P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stabiUty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW097P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide wiU be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophUicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, ldnking of the peptide chain by addition of proline, disulfide crossUnking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW097P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW097P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-teraύnus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teiminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW097P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutaπuc acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig.4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teπriinus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cisplatin is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

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Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cisplatin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW097P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbc<Uimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be tteated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW097P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alky 1 Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be hmited to the details shown. Rather,

15 CW097P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW097P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cisplatin covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein cisplatin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW097P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cisplatin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cisplatin from said composition in a pH-dependent manner.

19. A method for protecting cisplatin from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for conttolling release of cisplatin from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching cisplatin to said polypeptide.

21. A method for delivering cisplatin to a patient comprising administering to said patient a composition comprising: a polypeptide; and cisplatin covalently attached to said polypeptide.

22. The method of claim 21 wherein cisplatin is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein cisplatin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a poljφeptide and cisplatin covalentiy attached to the polypeptide. Also provided is a method for delivery of cisplatin to a patient comprising administering to the patient a composition comprising a polypeptide and cisplatin covalently attached to the polypeptide. Also provided is a method for protecting cisplatin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cisplatin from a composition comprising covalently attaching it to the polypeptide.

19 CW098P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CITALOPRAM AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to citalopram , as well as methods for protecting and administering citalopram . This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both weU smdied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Citalopram is a known pharmaceutical agent that is used in the treatment of depression. Its chemical name is l-[3-(dimethylamino)propyl]-l-(4-fluorophenyl)-l,3- dihydro-5-isobenzofurancarbonitrile. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW098P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, wUl assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW098P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of poljφeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW098P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (citalopram ) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching citalopram to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide wUl stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is conttoUed, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising citalopram microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and citalopram covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Citalopram preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW098P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting citalopram from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering citalopram to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, citalopram is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, citalopram is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and citalopram is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, citalopram is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, citalopram is released from the composition in a CW098P

sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching citalopram to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, citalopram and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is CW098P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize citalopram and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of citalopram . Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Citalopram is the subject of GB 1526331 (1978), GB 1486 (1976), and EP

171943 B (1988), herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises citalopram covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW098P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodjmamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW098P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide wiU be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophUicity of the polypeptide.

Ionizing amino acids can be selected for pH conttoUed peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but wiU ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW098P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW098P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or poljφeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide canier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW098P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide canier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW098P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in locaUzed delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-citalopram conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW098P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW098P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW098P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW098P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and citalopram covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturaUy occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein citalopram is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW098P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein citalopram is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing citalopram from said composition in a pH-dependent manner.

19. A method for protecting citalopram from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of citalopram from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching citalopram to said polypeptide.

21. A method for delivering citalopram to a patient comprising administering to said patient a composition comprising: a polypeptide; and citalopram covalently attached to said polypeptide.

22. The method of claim 21 wherein citalopram is released from said composition by an enzyme-catalyzed release.

18 CW098P

23. The method of claim 21 wherein citalopram is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and citalopram covalently attached to the polypeptide. Also provided is a method for delivery of citalopram to a patient comprising administering to the patient a composition comprising a polypeptide and citalopram covalently attached to the polypeptide. Also provided is a method for protecting citalopram from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of citalopram from a composition comprising covalently attaching it to the polypeptide.

19 CW099P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CLARITHROMYCIN

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalentiy attached to clarithromycin, as well as methods for protecting and administering clarithromycin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Clarithromycin is a known pharmaceutical agent that is used in the treatment of bacterial infection. Its chemical name is 6-O-methylerythromycin. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW099P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW099P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW099P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brash-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (clarithromycin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching clarithromycin to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising clarithromycin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and clarithromycin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Clarithromycin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW099P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting clarithromycin from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering clarithromycin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, clarithromycin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, clarithromycin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and clarithromycin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, clarithromycin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, clarithromycin is CW099P

released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching clarithromycin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, clarithromycin and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutaπuc acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW099P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize clarithromycin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of clarithromycin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also aUows targeted delivery of active agents to specifics sites of action.

Clarithromycin is the subject of EP 41355 B (1983) and EP 293885 B (1993), and

US apphcation number 58499 (1987), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises clarithromycin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopoljmier of one of the twenty naturally occurring amino acids, (iU) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and pol φeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW099P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalUne solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stabiUty, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are estabUshed during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stabiUty of the folded protein where the degree to which ideal packing occurs determines the degree of relative stabiUty of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW099P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodj amics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophUicity is desired, then the carrier polypeptide wiU be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophUicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but wUl ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW099P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table Usts the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW099P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutaπuc acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW099P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, clarithromycin is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW099P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetricaUy to cofactors. Thus, one can expect that excitation of the membrane transport system wiU involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-clarithromycin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW099P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the foUowing example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-teoninus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW099P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW099P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and clarithromycin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturaUy occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein clarithromycin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW099P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein clarithromycin is conformationaUy protected by folding of said poljφeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing clarithromycin from said composition in a pH-dependent manner.

19. A method for protecting clarithromycin from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of clarithromycin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching clarithromycin to said polypeptide.

21. A method for delivering clarithromycin to a patient comprising administering to said patient a composition comprising: a polypeptide; and clarithromycin covalently attached to said polypeptide.

22. The method of claim 21 wherein clarithromycin is released from said composition by an enzyme-catalyzed release.

17 CW099P

23. The method of claim 21 wherein clarithromycin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW099P

Abstract

A composition comprising a polypeptide and clarithromycin covalentiy attached to the polypeptide. Also provided is a method for delivery of clarithromycin to a patient comprising administering to the patient a composition comprising a polypeptide and clarithromycin covalently attached to the polypeptide. Also provided is a method for protecting clarithromycin from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of clarithromycin from a composition comprising covalently attaching it to the polypeptide.

19 CW100P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CLOMTPRAMINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to clomipramine, as well as methods for protecting and administering clomipramine. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Clomipramine is a known pharmaceutical agent that is used in the treatment of obsessive-compulsive disorder. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW100P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabUity are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabiUty of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings, incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CWIOOP

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CWIOOP

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (clomipramine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching clomipramine to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the poljφeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising clomipramine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and clomipramine covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Clomipramine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CWIOOP

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the poljφeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the poljφeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting clomipramine from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering clomipramine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, clomipramine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, clomipramine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and clomipramine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, clomipramine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, clomipramine is released CWIOOP

from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching clomipramine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) poljmierizing the active agent amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, clomipramine and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CWIOOP

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize clomipramine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of clomipramine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted deUvery of active agents to specifics sites of action.

The composition of the invention comprises clomipramine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (Ui) a heteropoljmier of two or more namrally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CWIOOP

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the soUd reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the sohd reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stabiUty of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CWIOOP

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CWIOOP

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug deUvery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CWIOOP

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the ohgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-teoninus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CWIOOP

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copoljmierization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CWIOOP

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epitheha efficientiy via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-clomipramine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CWIOOP

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with dusopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be freated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CWIOOP

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutaπuc acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CWIOOP

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and clomipramine covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein clomipramine is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CWIOOP

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein clomipramine is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing clomipramine from said composition in a pH-dependent manner.

19. A method for protecting clomipramine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of clomipramine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching clomipramine to said polypeptide.

21. A method for delivering clomipramine to a patient comprising administering to said patient a composition comprising: a polypeptide; and clomipramine covalently attached to said polypeptide.

22. The method of claim 21 wherein clomipramine is released from said composition by an enzyme-catalyzed release.

17 CWIOOP

23. The method of claim 21 wherein clomipramine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CWIOOP

Abstract

A composition comprising a polypeptide and clomipramine covalently attached to the polypeptide. Also provided is a method for delivery of cloimpramine to a patient comprising administering to the patient a composition comprising a polypeptide and clomipramine covalently attached to the polypeptide. Also provided is a method for protecting clomipramine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of clomipramine from a composition comprising covalentiy attaching it to the polypeptide.

19 CW101P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CLONAZEPAM AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to clonazepam, as weU as methods for protecting and administering clonazepam. This novel compound, refened to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Clonazepam is a known pharmaceutical agent that is used in the treatment of epilepsy. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability CW101P

of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent deUvery systems are often critical for the effective deUvery of a biologicaUy active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabUity are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in Ueu of an injection or another invasive technique. Increasing the stabiUty of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and ttansport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified CW101P

amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may compUcate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that CW101P

incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (clonazepam) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching clonazepam to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising clonazepam microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and clonazepam covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a CW101P

heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturaUy occurring amino acids and one or more synthetic amino acids.

Clonazepam preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting clonazepam from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering clonazepam to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, clonazepam is released from the composition by CW101P

an enzyme-catalyzed release. In another preferred embodiment, clonazepam is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and clonazepam is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, clonazepam is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, clonazepam is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching clonazepam to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, clonazepam and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a CW101P

carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabihze clonazepam and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of clonazepam. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also aUows targeted deUvery of active agents to specifics sites of action.

The composition of the invention comprises clonazepam covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture. CW101P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oU drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stabUity of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW101P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stabiUty of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophihcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW101P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly Hmited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin e (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW101P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxyhc acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampiciUin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW101P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not Hmited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, clonazepam is covalently attached to the polypeptide via the amino or nitro group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixmres of amino acid N-carboxyanhydrides.

12 CW101P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabUizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier poljφeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substrate. They include the amino acid, ohgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-clonazepam conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW101P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with dusopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be tteated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW101P

hydroxysuccirumide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystalUzed from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the πύxture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW101P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW101P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and clonazepam covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more sjmthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein clonazepam is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW101P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein clonazepam is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing clonazepam from said composition in a pH-dependent manner.

19. A method for protecting clonazepam from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of clonazepam from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching clonazepam to said polypeptide.

21. A method for delivering clonazepam to a patient comprising administering to said patient a composition comprising: a polypeptide; and clonazepam covalently attached to said polypeptide.

22. The method of claim 21 wherein clonazepam is released from said composition by an enzyme-catalyzed release.

18 CW101P

23. The method of claim 21 wherein clonazepam is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and clonazepam covalently attached to the polypeptide. Also provided is a method for delivery of clonazepam to a patient comprising administering to the patient a composition comprising a polypeptide and clonazepam covalently attached to the polypeptide. Also provided is a method for protecting clonazepam from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of clonazepam from a composition comprising covalentiy attaching it to the polypeptide.

19 CW102P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CLONIDINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to clonidine, as well as methods for protecting and administering clonidine. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Clonidine is a known pharmaceutical agent that is used in the treatment of hypertension. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW102P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibUity and perhaps even reduce the number of dosages required which could improve patient compUance.

Absoφtion of an oraUy adiriinistered active agent is often blocked by the harshly acidic stomach πulieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across Upid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW102P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typicaUy require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW102P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (clonidine) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching clonidine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide wUl stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising clonidine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and clonidine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Clonidine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW102P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terniinus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting clonidine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering clonidine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, clonidine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, clonidine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and clonidine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, clonidine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, clonidine is released from the composition in a sustained release. CW102P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching clonidine to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, clonidine and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is CW102P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize clonidine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of clonidine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises clonidine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring airiino acids, (Ui) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary structure and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW102P

protein interior is therefore more like a crystalline solid than an oU drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the sohd reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are estabUshed during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stabiUty of the folded protein where the degree to which ideal packing occurs determines the degree of relative stabiUty of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that Hpophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW102P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide wtil be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophUicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but wiU ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW102P

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the Ueum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the foUowing table tists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW102P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular ttansamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW102P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, clonidine is covalentiy attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW102P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-clonidine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW102P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW102P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the rnixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystaUized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW102P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and clonidine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturaUy occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein clonidine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW102P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein clonidine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing clonidine from said composition in a pH-dependent manner.

19. A method for protecting clonidine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of clonidine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching clonidine to said polypeptide.

21. A method for delivering clonidine to a patient comprising administering to said patient a composition comprising: a polypeptide; and clonidine covalently attached to said polypeptide.

22. The method of claim 21 wherein clonidine is released from said composition by an enzyme-catalyzed release.

17 CW102P

23. The method of claim 21 wherein clonidine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW102P

Abstract

A composition comprising a polypeptide and clonidine covalently attached to the polypeptide. Also provided is a method for delivery of clonidine to a patient comprising administering to the patient a composition comprising a polypeptide and clonidine covalently attached to the polypeptide. Also provided is a method for protecting clonidine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for conttolling release of clonidine from a composition comprising covalently attaching it to the poljφeptide.

19 CW103P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CLOPIDOGREL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to clopidogrel, as weU as methods for protecting and administering clopidogrel. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Clopidogrel is a known pharmaceutical agent that is used in the treatment of thrombosis and stroke. Its chemical name is (S)-alfa-(2-chlorophenyl)-6,7- d hydrothieno[3,2-c]pyridine-5(4H)-acetic acid methyl ester sulfate (1:1). Its stracture is:

O — S — 0 0

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW103P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabUity of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabiUty of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent dehvery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW103P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug deUvery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug deUvery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW103P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (clopidogrel) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching clopidogrel to the N-terrninus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide wiU stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising clopidogrel microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and clopidogrel covalently attached to the polypeptide. Preferably, the polypeptide is (i) an ohgopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Clopidogrel preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW103P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting clopidogrel from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering clopidogrel to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefeoed embodiment, clopidogrel is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, clopidogrel is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and clopidogrel is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, clopidogrel is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, clopidogrel is released from the composition in a CW103P

sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching clopidogrel to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, clopidogrel and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutaπuc acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW103P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabilize clopidogrel and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of clopidogrel. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Clopidogrel is the subject of U.S. Patent Numbers 4,529,596, 4,847,265, and

5,576,328, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises clopidogrel covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (Ui) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW103P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oU drop and so the best model for determining forces contributing to protein stability is the soUd reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stabUity of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW103P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophUicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but wUl ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW103P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug deUvery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the Ueum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Bό (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

LipophUic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW103P

delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus apphcation, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oUgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-teπninus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to polyφydroxypropylglutaπύne), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzjmiatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular ttansamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW103P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not Hmited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW103P

The addition of stabilizers to the composition has the potential of stabihzing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetricaUy to cofactors. Thus, one can expect that excitation of the membrane transport system wUl involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oUgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavaUability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-clopidogrel conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW103P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW103P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW103P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and clopidogrel covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oUgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturaUy occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein clopidogrel is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW103P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceuticaUy acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein clopidogrel is conformationaUy protected by folding of said polypeptide about said active agent.

18.^' The composition of claim 1 wherein said polypeptide is capable of releasing clopidogrel from said composition in a pH-dependent manner.

19. A method for protecting clopidogrel from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of clopidogrel from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching clopidogrel to said polypeptide.

21. A method for delivering clopidogrel to a patient comprising administering to said patient a composition comprising: a polypeptide; and clopidogrel covalently attached to said polypeptide.

22. The method of claim 21 wherein clopidogrel is released from said composition by an enzyme-catalyzed release.

17 CW103P

23. The method of claim 21 wherein clopidogrel is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW103P

Abstract

A composition comprising a polypeptide and clopidogrel covalently attached to the polypeptide. Also provided is a method for delivery of clopidogrel to a patient comprising administering to the patient a composition comprising a polypeptide and clopidogrel covalently attached to the polypeptide. Also provided is a method for protecting clopidogrel from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of clopidogrel from a composition comprising covalently attaching it to the polypeptide.

19 CW104P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING A SODIUM CHANNEL BLOCKER AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to a sodium channel blocker , as well as methods for protecting and administering a sodium channel blocker . This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

The sodium channel blocker of the present invention is a known pharmaceutical agent that is used in the treatment of pain. Its chemical name is (5R)-5-(2,3- d cMorophenyl)-6-(fluoromethyl)-2,4-pyriπιidinediamine. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW104P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabiUty of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW104P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW104P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brash-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (a sodium channel blocker ) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching a sodium channel blocker to the N-terminus, the C-terrninus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, deUvery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzjmiatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising a sodium channel blocker microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and a sodium channel blocker covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

A sodium channel blocker preferably is covalently attached to a side chain, the

N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached CW104P

to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the poljφeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-teiminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationaUy protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting a sodium channel blocker from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for deUvering a sodium channel blocker to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a preferred embodiment, a sodium channel blocker is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, a sodium channel blocker is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and a sodium channel blocker is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, a sodium channel blocker is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, a sodium channel blocker is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an CW104P

adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching a sodium channel blocker to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, a sodium channel blocker and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW104P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent deUvery. First, the invention can stabihze a sodium channel blocker and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of a sodium channel blocker . Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted deUvery of active agents to specifics sites of action.

A sodium channel blocker is the subject of WO 97/9317 (1997), herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises a sodium channel blocker covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the djmamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW104P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW104P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Upopbilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given appUcation. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW104P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. GeneraUy, it was only possible to load up to 1/10 of the total drug-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly Hmited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW104P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxyhc acid group of an active agent may be covalently attached to the N-terminus, the C-teπninus or the side chain of the oUgopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxyhc acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terrninus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to polyOiydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide canier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular ttansamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

11 CW104P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechamsm can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzjmies in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, a sodium channel blocker is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW104P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsicaUy asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system wiU involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-a sodium channel blocker conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW104P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodumide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW104P

hydroxysuccinimide. Examples of bases include pyπolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystalHzed from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW104P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW104P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and a sodium channel blocker covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oUgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturaUy occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturaUy occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein a sodium channel blocker is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW104P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceuticaUy acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein a sodium channel blocker is conformationally protected by folding of said polypeptide about said active agent

18. The composition of claim 1 wherein said polypeptide is capable of releasing a sodium channel blocker from said composition in a pH-dependent manner.

19. A method for protecting a sodium channel blocker from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for conttolling release of a sodium channel blocker from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching a sodium channel blocker to said polypeptide.

21. A method for delivering a sodium channel blocker to a patient comprising administering to said patient a composition comprising: a polypeptide; and a sodium channel blocker covalently attached to said polypeptide.

22. The method of claim 21 wherein a sodium channel blocker is released from said composition by an enzyme-catalyzed release.

18 CW104P

23. The method of claim 21 wherein a sodium channel blocker is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and a sodium channel blocker covalently attached to the polypeptide. Also provided is a method for delivery of a sodium channel blocker to a patient comprising administering to the patient a composition comprising a polypeptide and a sodium channel blocker covalently attached to the polypeptide. Also provided is a method for protecting a sodium channel blocker from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controUing release of a sodium channel blocker from a composition comprising covalently attaching it to the polypeptide.

19 CW001P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ABACAVIR SULFATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to abacavir sulfate, as well as methods for protecting and administering abacavir sulfate. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaimaceutical effectiveness.

BACKGROUND OF THE INVENTION

Abacavir sulfate is a known pharmaceutical agent - a carbocyclic 2'- deoxyguanosine nucleoside analogue that is a reverse ttanscriptase inhibitor used in the tteatment of HIV. Its chemical name is (lS,4R)-4-[2-amino-6-(cyclopropylamino)-9H- purin-9-yl]-2-cyclopentene-l -methanol. Its stracmre is as follows:

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absorbtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical CW001P compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW001P shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW001P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (abacavir sulfate) to a polymer of peptides or amino acids. The invention is distinguished from the above mentioned technologies by virtue of covalently attaching abacavir sulfate to the N- terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising abacavir microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and abacavir sulfate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW001P

abacavir sulfate preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting abacavir sulfate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering abacavir sulfate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, abacavir sulfate is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, abacavir sulfate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and abacavir sulfate is released from the composition by dissolution of the microencapsulating agent. In another prefened CW001P

embodiment, abacavir sulfate is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, abacavir sulfate is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching abacavir sulfate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, abacavir sulfate and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW001P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Apphcation Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize abacavir sulfate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of abacavir sulfate. Furthermore, active agents can be combined to produce synergistic effects.

Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Abacavir sulfate is the subject of U.S. Patent Numbers 5,034,394 and 5,089,500, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises abacavir sulfate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture. CW001P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW001P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same canier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an ohgopeptide. Eighteen was subtracted from the amino acid's

10 molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polyt Tosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, abacavir sulfate is covalently attached to the polypeptide via its alcohol group or, alternatively, its amino group.

The polypeptide canier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported substtate. They include the amino acid, ohgopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-abacavir sulfate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alky 1 Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CLAIMS

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and abacavir sulfate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein abacavir sulfate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein abacavir sulfate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing abacavir sulfate from said composition in a pH-dependent manner.

19. A method for protecting abacavir sulfate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of abacavir sulfate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching abacavir sulfate to said polypeptide.

21. A method for delivering abacavir sulfate to a patient comprising administering to said patient a composition comprising: a polypeptide; and abacavir sulfate covalently attached to said polypeptide.

22. The method of claim 21 wherein abacavir sulfate is released from said composition by an enzyme-catalyzed release. 23. The method of claim 21 wherein abacavir sulfate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and abacavir sulfate covalently attached to the polypeptide. Also provided is a method for delivery of abacavir sulfate to a patient comprising administering to the patient a composition comprising a polypeptide and abacavir sulfate covalently attached to the polypeptide. Also provided is a method for protecting abacavir sulfate from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of abacavir sulfate from a composition comprising covalently attaching it to a polypeptide.

19 CW002P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ABARELIX AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to abarelix, as well as methods for protecting and administering abarelix. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Abarelix is a known phaimaceutical agent that is used in the tteatment of prostate cancer, acting as a gonadottopin-releasing hormone antagonist. Its chemical name is N- acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L- seryl-N-methyl-L-tyrosyl-D-asparagynyl-L-N6-(l-methylethyl)-L-lysyl-L-prolyl-D- alaninamide. Abarelix is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is as follows:

CW002P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absorbtion; targeted delivery to particular tissue/ceU type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and ttansport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW002P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW002P

linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (abarelix) to a polymer of peptides or amino acids. The invention is distinguished from the above mentioned technologies by virtue of covalently attaching abarelix to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising abarelix microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and abarelix covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, CW002P

(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Abarelix preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting abarelix from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering abarelix to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the CW002P

polypeptide. In a prefened embodiment, abarelix is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, abarelix is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and abarelix is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, abarelix is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, abarelix is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching abarelix to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, abarelix and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a CW002P

carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize abarelix and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of abarelix. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises abarelix covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture. CW002P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW002P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW002P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW002P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide canier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly fooned free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW002P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, abarelix is covalently attached to the polypeptide via the free alcohol group or, alternatively, through one of its amino groups.

The polypeptide canier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW002P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-abarelix conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW002P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AciόVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW002P

hydroxysuccinimide. Examples of bases include pyoohdinop ridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW002P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW002P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and abarelix covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein abarelix is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW002P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein abarelix is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing abarelix from said composition in a pH-dependent manner.

19. A method for protecting abarelix from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of abarelix from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching abarelix to said polypeptide.

21. A method for delivering abarelix to a patient comprising administering to said patient a composition comprising: a polypeptide; and abarelix covalently attached to said polypeptide.

22. The method of claim 21 wherein abarelix is released from said composition by an enzyme-catalyzed release.

18 CW002P

23. The method of claim 21 wherein abarelix is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and abarelix covalently attached to the polypeptide. Also provided is a method for delivery of abarelix to a patient comprising administering to the patient a composition comprising a polypeptide and abarelix covalently attached to the polypeptide. Also provided is a method for protecting abarelix from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of abarelix from a composition comprising covalently attaching it to the polypeptide.

19 CW105P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CHLORPHENIRAMINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to chloφheniramine, as well as methods for protecting and administering chloφheniramine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a canier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Chloφheniramine is a known pharmaceutical agent that is used in the treatment of nasal congestion. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW.05P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW105P reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydrox φropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in ²⁰¹⁶ CW105P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (chloφheniramine tannate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching chloφheniramine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a canier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the canier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the canier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising chloφheniramine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and chloφheniramine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Chloφheniramine preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active ²⁰¹⁷ CW105P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting chloφheniramine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering chloφheniramine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, chloφheniramine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, chloφheniramine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and chloφheniramine is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, chloφheniramine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, chloφheniramine is ²⁰¹⁸ CW105P

released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching chloφheniramine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, chloφheniramine and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW105P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize chloφheniramine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of chloφheniramine tannate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises chloφheniramine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the ²⁰²⁰ CW105P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's ²⁰²¹ CW105P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the canier polypeptide will be emiched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of CW105P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular canier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW105P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 ²⁰²⁴ CW105P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide caoier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW105P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-chloφheniramine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 ²⁰²⁶ CW105P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 ²⁰²⁷ CW105P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW105P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and chloφheniramine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein chloφheniramine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW105P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein chloφheniramine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing chloφheniramine from said composition in a pH-dependent manner.

19. A method for protecting chloφheniramine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of chloφheniramine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching chloφheniramine to said poljφeptide.

21. A method for delivering chloφheniramine to a patient comprising administering to said patient a composition comprising: a polypeptide; and chloφheniramine covalently attached to said polypeptide.

22. The method of claim 21 wherein chloφheniramine is released from said composition by an enzyme-catalyzed release.

17 CW105P

23. The method of claim 21 wherein chloφheniramine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW105P

Abstract

A composition comprising a polypeptide and chloφheniramine covalently attached to the polypeptide. Also provided is a method for delivery of chloφheniramine to a patient comprising administering to the patient a composition comprising a poljφeptide and chloφheniramine covalently attached to the polypeptide. Also provided is a method for protecting chloφheniramine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of chloφheniramine from a composition comprising covalently attaching it to the polypeptide.

CW106P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CLOZAPINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to clozapine, as well as methods for protecting and administering clozapine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Clozapine is a known pharmaceutical agent that^" is used in the treatment of psychotic disorders. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; CW106P

and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations. CW106P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the garnma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. ²⁰³⁵ CW106P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caniers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (clozapine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching clozapine to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a canier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising clozapine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and clozapine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a ²⁰³⁶ CW106P

heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Clozapine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting clozapine from degradation comprising covalently attaching it to a poljφeptide.

The invention also provides a method for delivering clozapine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, clozapine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, clozapine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. CW106P

In another prefeoed embodiment, the composition further comprises a microencapsulating agent and clozapine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, clozapine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, clozapine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching clozapine to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, clozapine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. ²⁰³⁸ CW106P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize clozapine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of clozapine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises clozapine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuning amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW106P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW106P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of CW106P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 ²⁰⁴² CW106P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using ²⁰⁴³ CW106P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, clozapine is covalently attached to the polypeptide via the amino group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW106P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-clozapine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 ^{20 5} CW106P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

AIcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC,

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 ²⁰⁴⁶ CW106P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 ²⁰⁴⁷ CW106P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and clozapine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein clozapine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW106P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein clozapine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing clozapine from said composition in a pH-dependent manner.

19. A method for protecting clozapine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of clozapine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching clozapine to said polypeptide.

21. A method for delivering clozapine to a patient comprising administering to said patient a composition comprising: a polypeptide; and clozapine covalently attached to said polypeptide.

22. The method of claim 21 wherein clozapine is released from said composition by an enzyme-catalyzed release.

17 CW106P

23. The method of claim 21 wherein clozapine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

²⁰⁵⁰ CW106P

Abstract

A composition comprising a polypeptide and clozapine covalently attached to the polypeptide. Also provided is a method for delivery of clozapine to a patient comprising administering to the patient a composition comprising a polypeptide and clozapine covalently attached to the polypeptide. Also provided is a method for protecting clozapine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of clozapine from a composition comprising covalently attaching it to the polypeptide.

19 ²⁰⁵¹ CW107P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING COLESTIPOL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to colestipol, as well as methods for protecting and administering colestipol. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaimaceutical effectiveness.

BACKGROUND OF THE INVENTION

Colestipol is a known pharmaceutical agent that is used in the treatment of hypercholesterolemia. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Colestipol hydrochloride is a copolymer of diethylenetriamine and l-chloro-2,3-epoxypropane that contains secondary and tertiary amines with approximately 1 out of 5 amine nitrogens protonated with chloride.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken CW107P

under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet fo nulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent ²⁰⁵³ CW107P

in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR ²⁰⁵⁴ CW107P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (colestipol) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching colestipol to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising colestipol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and colestipol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occuning amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Colestipol preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is ²⁰⁵⁵ CW107P

an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting colestipol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering colestipol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, colestipol is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, colestipol is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and colestipol is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, colestipol is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, colestipol is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is ²⁰⁵⁶ CW107P

controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching colestipol to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, colestipol and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW107P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize colestipol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of colestipol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises colestipol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. ²⁰⁵⁸ CW107P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be ²⁰⁵⁹ CW107P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the canier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular canier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate 2060 CW107P

weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 ²⁰⁶¹ CW107P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 ²⁰⁶² CW107P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This canier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, colestipol is covalently attached to the polypeptide via one of its amino groups.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW107P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-colestipol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide canier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW107P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

AIcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-AIkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW107P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW107P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and colestipol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuning amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuning amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein colestipol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 ²⁰⁶⁷ CW107P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein colestipol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing colestipol from said composition in a pH-dependent manner.

19. A method for protecting colestipol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of colestipol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching colestipol to said polypeptide.

21. A method for delivering colestipol to a patient comprising administering to said patient a composition comprising: a polypeptide; and colestipol covalently attached to said polypeptide.

22. The method of claim 21 wherein colestipol is released from said composition by an enzyme-catalyzed release.

17 CW107P

23. The method of claim 21 wherein colestipol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW107P

Abstract

A composition comprising a polypeptide and colestipol covalently attached to the polypeptide. Also provided is a method for delivery of colestipol to a patient comprising administering to the patient a composition comprising a polypeptide and colestipol covalently attached to the polypeptide. Also provided is a method for protecting colestipol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of colestipol from a composition comprising covalently attaching it to the polypeptide.

19 CW108P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CONIVAPTAN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to conivaptan, as well as methods for protecting and administering conivaptan. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Conivaptan is a known pharmaceutical agent that is used in the treatment of congestive heart failure and hyponatremia. Its chemical name is N-[l,l'-biphenyl]-2-yl- 4-[(4,5-dihydro-2-methylimidazo[4,5-d][l]benzazepin-6(lH)-yl)carbonyl]-benzamide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical CW108P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW108P

shortcomings, incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW108P

diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (conivaptan) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching conivaptan to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the canier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising conivaptan microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and conivaptan covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids. CW108P

Conivaptan preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In * another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- te ninus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting conivaptan from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering conivaptan to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, conivaptan is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, conivaptan is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and conivaptan is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, conivaptan is CW108P

released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, conivaptan is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching conivaptan to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, conivaptan and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW108P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize conivaptan and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of conivaptan. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Conivaptan is the subject of EP 709386 A (1996), herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises conivaptan covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure. CW108P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW108P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the canier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW108P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW108P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW108P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

12 CW108P

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

13 CW108P

Preferably, the resultant peptide-conivaptan conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide canier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide canier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated

14 CW108P

solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide canier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

15 CW108P

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW108P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and conivaptan covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuning amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein conivaptan is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW108P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein conivaptan is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing conivaptan from said composition in a pH-dependent manner.

19. A method for protecting conivaptan from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of conivaptan from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching conivaptan to said polypeptide.

21. A method for delivering conivaptan to a patient comprising administering to said patient a composition comprising: a polypeptide; and conivaptan covalently attached to said polypeptide.

22. The method of claim 21 wherein conivaptan is released from said composition by an enzyme-catalyzed release. CW108P

23. The method of claim 21 wherein conivaptan is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and conivaptan covalently attached to the polypeptide. Also provided is a method for delivery of conivaptan to a patient comprising administering to the patient a composition comprising a polypeptide and conivaptan covalently attached to the polypeptide. Also provided is a method for protecting conivaptan from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of conivaptan from a composition comprising covalently attaching it to the polypeptide.

19 CW109P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CYCLOBENZAPRINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cyclobenzaprine, as well as methods for protecting and administering cyclobenzaprine. This novel compound, refeoed to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cyclobenzaprine is a known pharmaceutical agent that is used in the treatment of muscle spasm. Its chemical name is 3-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-N,N- dimethyl-1 -propanamine. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW109P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW109P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caniers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW109P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cyclobenzaprine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cyclobenzaprine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cyclobenzaprine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cyclobenzaprine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Cyclobenzaprine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW109P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cyclobenzaprine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cyclobenzaprine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, cyclobenzaprine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, cyclobenzaprine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and cyclobenzaprine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cyclobenzaprine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cyclobenzaprine is CW109P

released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cyclobenzaprine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cyclobenzaprine and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW109P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cyclobenzaprine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cyclobenzaprine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cyclobenzaprine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW109P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW109P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW109P

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Tbuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant CW109P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide canier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW109P

maximum drug loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This canier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide caoier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW109P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cyclobenzaprine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW109P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

AIcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide canier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or fributylamine.

14 CW109P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl GIutamate7C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention. CW109P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cyclobenzaprine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cyclobenzaprine is covalently attached to a side chain, the N-terminus or the C-terminus of said poljφeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW109P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cyclobenzaprine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cyclobenzaprine from said composition in a pH-dependent manner.

19. A method for protecting cyclobenzaprine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cyclobenzaprine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cyclobenzaprine to said polypeptide.

21. A method for delivering cyclobenzaprine to a patient comprising administering to said patient a composition comprising: a polypeptide; and cyclobenzaprine covalently attached to said polypeptide.

22. The method of claim 21 wherein cyclobenzaprine is released from said composition by an enzyme-catalyzed release.

17 CW109P

23. The method of claim 21 wherein cyclobenzaprine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW109P

Abstract

A composition comprising a polypeptide and cyclobenzaprine covalently attached to the polypeptide. Also provided is a method for delivery of cyclobenzaprine to a patient comprising administering to the patient a composition comprising a polypeptide and cyclobenzaprine covalently attached to the polypeptide. Also provided is a method for protecting cyclobenzaprine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cyclobenzaprine from a composition comprising covalently attaching it to the polypeptide.

19 CW110P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CYCLOPHOSPHAMIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cyclophosphamide, as well as methods for protecting and administering cyclophosphamide. This novel compound, refeoed to as a CARRTERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cyclophosphamide is a known pharmaceutical agent that is used in the treatment of myeloproliferative and lymphoproliferative disorders and solid malignancies. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW110P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW110P

reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW110P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cyclophosphamide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cyclophosphamide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cyclophosphamide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cyclophosphamide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuning amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Cyclophosphamide preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active CW110P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cyclophosphamide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cyclophosphamide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cyclophosphamide is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cyclophosphamide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and cyclophosphamide is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cyclophosphamide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cyclophosphamide is CW110P

released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cyclophosphamide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cyclophosphamide and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW110P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cyclophosphamide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cyclophosphamide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cyclophosphamide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW110P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW110P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same canier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the canier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the canier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW110P

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW110P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW110P

maximum drug loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cyclophosphamide is covalently attached to the polypeptide via the amino group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW110P

invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cyclophosphamide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW110P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW110P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW110P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cyclophosphamide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cyclophosphamide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW110P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cyclophosphamide is conformationally protected by folding of said poljφeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cyclophosphamide from said composition in a pH-dependent manner.

19. A method for protecting cyclophosphamide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cyclophosphamide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cyclophosphamide to said polypeptide.

21. A method for delivering cyclophosphamide to a patient comprising administering to said patient a composition comprising: a polypeptide; and cyclophosphamide covalently attached to said polypeptide.

22. The method of claim 21 wherein cyclophosphamide is released from said composition by an enzyme-catalyzed release.

17 CW110P

23. The method of claim 21 wherein cyclophosphamide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW110P

Abstract

A composition comprising a polypeptide and cyclophosphamide covalently attached to the polypeptide. Also provided is a method for delivery of cyclophosphamide to a patient comprising administering to the patient a composition comprising a polypeptide and cyclophosphamide covalently attached to the polypeptide. Also provided is a method for protecting cyclophosphamide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cyclophosphamide from a composition comprising covalently attaching it to the polypeptide.

19 CW111P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING CYCLOSPORINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cyclosporine, as well as methods for protecting and administering cyclosporine. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cyclosporine is a known pharmaceutical agent that is used in the treatment of prevention of rejection of kidney, liver or heart allografts. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW111P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW111P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW111P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (cyclosporine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cyclosporine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising cyclosporine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cyclosporine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Cyclosporine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW111P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cyclosporine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cyclosporine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cyclosporine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cyclosporine is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and cyclosporine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, cyclosporine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cyclosporine is released from the CW111P

composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cyclosporine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cyclosporine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW111P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cyclosporine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cyclosporine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises cyclosporine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial arrangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW111P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW111P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW111P

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW111P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW111P

maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cyclosporine is covalently attached to the polypeptide via the hydroxyl group or, alternatively, via an artificial linker.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW111P

invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-cyclosporine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW111P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW111P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-AIkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW111P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cyclosporine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuning amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cyclosporine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW111P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cyclosporine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cyclosporine from said composition in a pH-dependent manner.

19. A method for protecting cyclosporine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cyclosporine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cyclosporine to said polypeptide.

21. A method for delivering cyclosporine to a patient comprising administering to said patient a composition comprising: a polypeptide; and cyclosporine covalently attached to said polypeptide.

22. The method of claim 21 wherein cyclosporine is released from said composition by an enzyme-catalyzed release.

17 CW111P

23. The method of claim 21 wherein cyclosporine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW111P

Abstract

A composition comprising a polypeptide and cyclosporine covalently attached to the polypeptide. Also provided is a method for delivery of cyclosporine to a patient comprising administering to the patient a composition comprising a polypeptide and cyclosporine covalently attached to the polypeptide. Also provided is a method for protecting cyclosporine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cyclosporine from a composition comprising covalently attaching it to the polypeptide.

19 CW112P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DALTEPARIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to dalteparin, as well as methods for protecting and administering dalteparin. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Dalteparin (also known as heparin) is a known pharmaceutical agent that is used in the treatment of prevention of ischemic complications, due to blood clot formation in patients with unstable angina and non-Q-wave myocardial infarction receiving concuoent aspirin therapy. It is a natural product that is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase CW112P

markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some CW112P

technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR CW112P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (dalteparin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching dalteparin to the N- terminus, the C-teoninus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising dalteparin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and dalteparin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Dalteparin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is CW112P

an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting dalteparin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering dalteparin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, dalteparin is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, dalteparin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and dalteparin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, dalteparin is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, dalteparin is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW112P

controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching dalteparin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, dalteparin and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW112P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize dalteparin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of dalteparin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Dalteparin is the subject of EP 14184 B (1989), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises dalteparin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW112P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW112P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW112P

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus apphcation, for those active agents attached to pendant

10 CW112P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW112P

maximum drug loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, dalteparin is covalently attached to the polypeptide via any free hydroxyl, amino, or carboxyl group or, alternatively, via an artificial linker.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW112P

invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-dalteparin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW112P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW112P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW112P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and dalteparin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuning amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein dalteparin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW112P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein dalteparin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing dalteparin from said composition in a pH-dependent manner.

19. A method for protecting dalteparin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of dalteparin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching dalteparin to said polypeptide.

21. A method for delivering dalteparin to a patient comprising administering to said patient a composition comprising: a polypeptide; and dalteparin covalently attached to said polypeptide.

22. The method of claim 21 wherein dalteparin is released from said composition by an enzyme-catalyzed release.

17 CW112P

23. The method of claim 21 wherein dalteparin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW112P

Abstract

A composition comprising a polypeptide and dalteparin covalently attached to the polypeptide. Also provided is a method for delivery of dalteparin to a patient comprising administering to the patient a composition comprising a polypeptide and dalteparin covalently attached to the polypeptide. Also provided is a method for protecting dalteparin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of dalteparin from a composition comprising covalently attaching it to the polypeptide.

19 CW113P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DAPITANT AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to dapitant, as well as methods for protecting and administering dapitant. This novel compound, refeoed to ΉS a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Dapitant is a known pharmaceutical agent that is used in the treatment of asthma. Its chemical name is [3aS-[2(R*),3aalpha,4beta,7aalpha]]-octahydro-4-(2- methoxyphenyl)-2-[2-(2-methoxyphenyl)-l-oxopropyl]-7,7-diphenyl-lH-isoindol-4-ol. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW113P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW113P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW113P

diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (dapitant) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching dapitant to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the canier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising dapitant microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and dapitant covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW113P

Dapitant preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting dapitant from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering dapitant to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, dapitant is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, dapitant is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and dapitant is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, dapitant is released CW113P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, dapitant is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching dapitant to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, dapitant and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW113P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize dapitant and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of dapitant. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Dapitant is the subject of WO 93/21155 (1993), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises dapitant covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occuning amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW113P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW113P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW113P

caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular CW113P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

11 CW113P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, dapitant is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW113P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-dapitant conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW113P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW113P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW113P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and dapitant covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein dapitant is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW113P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein dapitant is conformationally protected by folding of said poljφeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing dapitant from said composition in a pH-dependent manner.

19. A method for protecting dapitant from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of dapitant from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching dapitant to said polypeptide.

21. A method for delivering dapitant to a patient comprising administering to said patient a composition comprising: a polypeptide; and dapitant covalently attached to said polypeptide.

22. The method of claim 21 wherein dapitant is released from said composition by an enzyme-catalyzed release.

17 CW113P

23. The method of claim 21 wherein dapitant is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW113P

Abstract

A composition comprising a polypeptide and dapitant covalently attached to the polypeptide. Also provided is a method for delivery of dapitant to a patient comprising administering to the patient a composition comprising a polypeptide and dapitant covalently attached to the polypeptide. Also provided is a method for protecting dapitant from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of dapitant from a composition comprising covalently attaching it to the polypeptide.

19 CW114P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DESMOPRESSIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to desmopressin, as well as methods for protecting and administering desmopressin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Desmopressin is a known pharmaceutical agent that is used in the treatment of urinary incontinence. Its chemical name is l-(3-mercaptopropanic acid)-8-D-arginine- vasopressin. Its stracture is:

S ICHjCKi_C ϊ-Tyf-Plι«-βlϊS-A«S-Cτ IS-P'9-(t-Arg-6l)|-MHa - CH₃OQ0W

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase CW114P

markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some CW114P

technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR CW114P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (desmopressin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching desmopressin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising desmopressin microencapsulated by a poljφeptide.

The invention provides a composition comprising a polypeptide and desmopressin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Desmopressin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is CW114P

an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting desmopressin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering desmopressin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, desmopressin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, desmopressin is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and desmopressin is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, desmopressin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, desmopressin is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The CW114P

adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching desmopressin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino, acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, desmopressin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW114P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize desmopressin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of desmopressin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Desmopressin is the subject of U.S. Patent Numbers 5,047,398, 5,500,413, 5,674,850, and 5,763,407, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises desmopressin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the CW114P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW114P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of CW114P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent CW114P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW114P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This canier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus t φe of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, desmopressin is covalently attached to the polypeptide via an amide linkage with the amino group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW114P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-desmopressin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW114P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide canier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide canier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW114P

Preparation of γ-AIkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW114P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and desmopressin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuning amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein desmopressin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW114P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein desmopressin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing desmopressin from said composition in a pH-dependent manner.

19. A method for protecting desmopressin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of desmopressin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching desmopressin to said polypeptide.

21. A method for delivering desmopressin to a patient comprising administering to said patient a composition comprising: a polypeptide; and desmopressin covalently attached to said polypeptide.

22. The method of claim 21 wherein desmopressin is released from said composition by an enzyme-catalyzed release.

17 CW114P

23. The method of claim 21 wherein desmopressin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW114P

Abstract

A composition comprising a polypeptide and desmopressin covalently attached to the polypeptide. Also provided is a method for delivery of desmopressin to a patient comprising administering to the patient a composition comprising a polypeptide and desmopressin covalently attached to the polypeptide. Also provided is a method for protecting desmopressin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of desmopressin from a composition comprising covalently attaching it to the polypeptide.

19 CW115P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DESOGESTREL

AND ETHINYL ESTRADIOL AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to desogestrel and ethinyl estradiol, as well as methods for protecting and administering desogestrel and ethinyl estradiol. This novel compound, refened to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Desogestrel and ethinyl estradiol are known pharmaceutical agents used together as a contraceptive.

Desogestrel's chemical name is (17alpha)-13-ethyl-l l-methylene-18,19- dinoφregn-4-en-20-yn-17-ol. Its structure is:

Ethinyl estradiol' s chemical name is (17alpha)-19-noφregna-l,2,5(10)-trien-20- yne-3,17-diol. Its stracture is:

CW115P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW115P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW115P

linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (desogestrel and ethinyl estradiol) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching desogestrel and ethinyl estradiol to the N-terminus, the C-teraiinus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising desogestrel and ethinyl estradiol microencapsulated by a polypeptide. CW115P

The invention provides a composition comprising a polypeptide and desogestrel and ethinyl estradiol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Desogestrel and ethinyl estradiol preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting desogestrel and ethinyl estradiol from degradation comprising covalently attaching it to a polypeptide. CW115P

The invention also provides a method for delivering desogestrel and ethinyl estradiol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, desogestrel and ethinyl estradiol are released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, desogestrel and ethinyl estradiol are released in a time- dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and desogestrel and ethinyl estradiol are released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, desogestrel and ethinyl estradiol are released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, desogestrel and ethinyl estradiol are released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching desogestrel and ethinyl estradiol to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, desogestrel and ethinyl estradiol and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the CW115P

polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize desogestrel and ethinyl estradiol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of desogestrel and ethinyl estradiol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Desogestrel are the subject of GB 1455270 (1976), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises desogestrel and ethinyl estradiol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW115P

heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of CW115P

maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of CW115P

the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the canier poljφeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW115P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

11 CW115P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide canier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide canier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, poljftyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW115P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, desogestrel and ethinyl estradiol are covalently attached to the polypeptide via their hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the canier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW115P

mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-desogestrel and ethinyl estradiol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

14 CW115P

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW115P

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW115P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and desogestrel and ethinyl estradiol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuning amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuning amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein desogestrel and ethinyl estradiol are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW115P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein desogestrel and ethinyl estradiol are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing desogestrel and ethinyl estradiol from said composition in a pH-dependent manner.

19. A method for protecting desogestrel and ethinyl estradiol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of desogestrel and ethinyl estradiol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching desogestrel and ethinyl estradiol to said polypeptide.

21. A method for delivering desogestrel and ethinyl estradiol to a patient comprising administering to said patient a composition comprising: a polypeptide; and desogestrel and ethinyl estradiol covalently attached to said polypeptide.

22. The method of claim 21 wherein desogestrel and ethinyl estradiol are released from said composition by an enzyme-catalyzed release.

18 CW115P

23. The method of claim 21 wherein desogestrel and ethinyl estradiol are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and desogestrel and ethinyl estradiol covalently attached to the polypeptide. Also provided is a method for delivery of desogestrel and ethinyl estradiol to a patient comprising administering to the patient a composition comprising a polypeptide and desogestrel and ethinyl estradiol covalently attached to the polypeptide. Also provided is a method for protecting desogestrel and ethinyl estradiol from degradation comprising covalently attaching it to a poljφeptide. Also provided is a method for controlling release of desogestrel and ethinyl estradiol from a composition comprising covalently attaching it to the polypeptide.

19 CW116P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DEXTROAMPHETAMINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to dextroamphetamine, as well as methods for protecting and administering dextroamphetamine. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Dextroamphetamine is a known pharmaceutical agent that is used in the treatment of narcolepsy and attention deficit hyperactivity disorder. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW116P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW116P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW116P

diffusion rates and pharmacokinetics. High molecular weight caniers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (dextroamphetamine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching dextroamphetamine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising dextroamphetamine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and dextroamphetamine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW116P

Dextroamphetamine preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting dextroamphetamine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering dextroamphetamine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, dextroamphetamine is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, dextroamphetamine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and dextroamphetamine is released from the composition by dissolution of the microencapsulating agent. In another CW116P

prefeoed embodiment, dextroamphetamine is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, dextroamphetamine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching dextroamphetamine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, dextroamphetamine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW116P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize dextroamphetamine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of dextroamphetamine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises dextroamphetamine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW116P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW116P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of CW116P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an ohgopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW116P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW116P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This canier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus t φe of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, dextroamphetamine is covalently attached to the polypeptide via the amino group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW116P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-dextroamphetamine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW116P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide canier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW116P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW116P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and dextroamphetamine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein dextroamphetamine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW116P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein dextroamphetamine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing dextroamphetamine from said composition in a pH-dependent manner.

19. A method for protecting dextroamphetamine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of dextroamphetamine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching dextroamphetamine to said polypeptide.

21. A method for delivering dextroamphetamine to a patient comprising administering to said patient a composition comprising: a polypeptide; and dextroamphetamine covalently attached to said polypeptide.

22. The method of claim 21 wherein dextroamphetamine is released from said composition by an enzyme-catalyzed release.

17 ²²³⁹ CW116P

23. The method of claim 21 wherein dextroamphetamine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 ²²⁴⁰ CW116P

Abstract

A composition comprising a polypeptide and dextroamphetamine covalently attached to the polypeptide. Also provided is a method for delivery of dextroamphetamine to a patient comprising administering to the patient a composition comprising a polypeptide and dextroamphetamine covalently attached to the polypeptide. Also provided is a method for protecting dextroamphetamine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of dextroamphetamine from a composition comprising covalently attaching it to the polypeptide.

19 CW117P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DEXTROMETHORPHAN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to dextromethoφhan, as well as methods for protecting and administering dextromethoφhan. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Dextromethoφhan is a known pharmaceutical agent that is used in the treatment of coughs. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW117P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW117P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW117P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brash-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (dextromethoφhan) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching dextromethoφhan to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising dextromethoφhan microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and dextromethoφhan covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Dextromethoφhan preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active CW117P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting dextromethoφhan from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering dextromethoφhan to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, dextromethoφhan is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, dextromethoφhan is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and dextromethoφhan is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, dextromethoφhan is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, dextromethoφhan is CW117P

released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching dextromethorphan to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, dextromethoφhan and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW117P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize dextromethoφhan and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of dextromethoφhan. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises dextromethoφhan covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW117P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of irreversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW117P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the canier peptide and the active agent. CW117P

Dextran is the only polysaccharide known that has been explored as a macromolecular canier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW117P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly fooned free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW117P

maximum drug loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 ²²⁵³ CW117P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-dextromethoφhan conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW117P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

AIcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoohdinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW117P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 ²²⁵⁶ CW117P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and dextromethoφhan covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein dextromethoφhan is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW117P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein dextromethoφhan is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing dextromethoφhan from said composition in a pH-dependent manner.

19. A method for protecting dextromethoφhan from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of dextromethoφhan from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching dextromethoφhan to said polypeptide.

21. A method for delivering dextromethoφhan to a patient comprising administering to said patient a composition comprising: a polypeptide; and dextromethoφhan covalently attached to said polypeptide.

22. The method of claim 21 wherein dextromethoφhan is released from said composition by an enzyme-catalyzed release.

17 ²²⁵⁸ CW117P

23. The method of claim 21 wherein dextromethoφhan is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW117P

Abstract

A composition comprising a polypeptide and dextromethoφhan covalently attached to the polypeptide. Also provided is a method for delivery of dextromethoφhan to a patient comprising administering to the patient a composition comprising a polypeptide and dextromethoφhan covalently attached to the polypeptide. Also provided is a method for protecting dextromethoφhan from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of dextromethoφhan from a composition comprising covalently attaching it to the polypeptide.

19 CW118P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DIAZEPAM AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to diazepam, as well as methods for protecting and administering diazepam. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Diazepam is a known phaonaceutical agent that is used in the treatment of anxiety. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

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The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW118P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW118P

linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caniers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (diazepam) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching diazepam to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising diazepam microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and diazepam covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, CW118P

(ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Diazepam preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting diazepam from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering diazepam to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the ²²⁶⁵ CW118P

polypeptide. In a prefeoed embodiment, diazepam is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, diazepam is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and diazepam is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, diazepam is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, diazepam is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching diazepam to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, diazepam and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a CW118P

carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize diazepam and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of diazepam. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises diazepam covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure. CW118P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW118P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the canier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW118P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

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molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide canier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW118P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This canier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

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The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier poljφeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

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Preferably, the resultant peptide-diazepam conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide canier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated

14 CW118P

solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

15 CW118P

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW118P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and diazepam covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein diazepam is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW118P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein diazepam is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing diazepam from said composition in a pH-dependent manner.

19. A method for protecting diazepam from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of diazepam from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching diazepam to said polypeptide.

21. A method for delivering diazepam to a patient comprising administering to said patient a composition comprising: a polypeptide; and diazepam covalently attached to said polypeptide.

22. The method of claim 21 wherein diazepam is released from said composition by an enzyme-catalyzed release. CW118P

23. The method of claim 21 wherein diazepam is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and diazepam covalently attached to the polypeptide. Also provided is a method for delivery of diazepam to a patient comprising administering to the patient a composition comprising a polypeptide and diazepam covalently attached to the polypeptide. Also provided is a method for protecting diazepam from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of diazepam from a composition comprising covalently attaching it to the polypeptide.

19 CW119P

A NOVEL PHARMACEUTICAL COMPOUND AND METHODS OF MAKING

AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to cholinergic channel modulator, as well as methods for protecting and administering cholinergic channel modulator. This novel compound, refeoed to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Cholinergic channel modulator is a known phaonaceutical agent that is used in the treatment of pain. Its chemical name is (R)-2-chloro-5-(2- azetidinylmethoxy)pyridine. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW119P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW119P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caniers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW119P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(cholinergic channel modulator) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching cholinergic channel modulator to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising cholinergic channel modulator microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and cholinergic channel modulator covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuning amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Cholinergic channel modulator preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the CW119P

polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting cholinergic channel modulator from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering cholinergic channel modulator to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, cholinergic channel modulator is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, cholinergic channel modulator is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and cholinergic channel modulator is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, cholinergic channel modulator is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, cholinergic channel modulator is released CW119P

from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching cholinergic channel modulator to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, cholinergic channel modulator and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW119P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize cholinergic channel modulator and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of cholinergic channel modulator. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Cholinergic channel modulator is the subject of WO 96/40682 (1996), based on priority US application 474873, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises cholinergic channel modulator covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure. CW119P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of irreversible chemical or conformation processes. CW119P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the canier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the canier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW119P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the canier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW119P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW119P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, cholinergic channel modulator is covalently attached to the polypeptide via the amine group.

The polypeptide caoier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW119P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

13 CW119P

Preferably, the resultant peptide-cholinergic channel modulator conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

14 CW119P

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically

15 CW119P

overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW119P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and cholinergic channel modulator covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein cholinergic channel modulator is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW119P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein cholinergic channel modulator is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing cholinergic channel modulator from said composition in a pH-dependent manner.

19. A method for protecting cholinergic channel modulator from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of cholinergic channel modulator from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching cholinergic channel modulator to said polypeptide.

21. A method for delivering cholinergic channel modulator to a patient comprising administering to said patient a composition comprising: a polypeptide; and cholinergic channel modulator covalently attached to said polypeptide.

22. The method of claim 21 wherein cholinergic channel modulator is released from said composition by an enzyme-catalyzed release.

18 CW119P

23. The method of claim 21 wherein cholinergic channel modulator is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said poljφeptide.

Abstract

A composition comprising a polypeptide and cholinergic channel modulator covalently attached to the polypeptide. Also provided is a method for delivery of cholinergic channel modulator to a patient comprising administering to the patient a composition comprising a polypeptide and cholinergic channel modulator covalently attached to the polypeptide. Also provided is a method for protecting cholinergic channel modulator from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of cholinergic channel modulator from a composition comprising covalently attaching it to the polypeptide.

19 CW120P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DICLOFENAC AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to diclofenac, as well as methods for protecting and administering diclofenac. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Diclofenac is a known pharmaceutical agent that is used in the treatment of acute and chronic rheumatoid arthritis. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its chemical name is potassium (o-(2,6-dichloroanilino)-phenyl)acetate. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered CW120P

product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations. CW120P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW120P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (diclofenac) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching diclofenac to the N- teoninus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising diclofenac microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and diclofenac covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW120P

heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Diclofenac preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting diclofenac from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering diclofenac to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, diclofenac is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, diclofenac is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed CW120P

release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and diclofenac is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, diclofenac is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, diclofenac is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching diclofenac to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, diclofenac and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW120P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize diclofenac and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of diclofenac. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises diclofenac covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW120P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW120P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conforrhational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of CW120P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW120P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW120P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This canier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, diclofenac is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW120P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-diclofenac conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW120P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide canier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW120P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW120P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and diclofenac covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein diclofenac is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW120P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein diclofenac is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing diclofenac from said composition in a pH-dependent manner.

19. A method for protecting diclofenac from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of diclofenac from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching diclofenac to said polypeptide.

21. A method for delivering diclofenac to a patient comprising administering to said patient a composition comprising: a polypeptide; and diclofenac covalently attached to said polypeptide.

22. The method of claim 21 wherein diclofenac is released from said composition by an enzyme-catalyzed release.

17 CW120P

23. The method of claim 21 wherein diclofenac is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

²³¹⁶ CW120P

Abstract

A composition comprising a polypeptide and diclofenac covalently attached to the polypeptide. Also provided is a method for delivery of diclofenac to a patient comprising administering to the patient a composition comprising a polypeptide and diclofenac covalently attached to the polypeptide. Also provided is a method for protecting diclofenac from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of diclofenac from a composition comprising covalently attaching it to the polypeptide.

19 CW121P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DICLOFENAC AND MISOPROSTOL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to diclofenac and misoprostol, as well as methods for protecting and administering diclofenac and misoprostol. This novel compound, refened to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its phaimaceutical effectiveness.

BACKGROUND OF THE INVENTION

Diclofenac and misoprostol are known pharmaceutical agents that are used together in the treatment of pain and inflammation. Each is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The chemical name of diclofenac is potassium (o-(2,6-dichloroanilino)- phenyl)acetate. Its structure is:

0 II

CW121P

The stracture of misoprostol is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme CW121P

degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foirnulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide CW121P

spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (diclofenac and misoprostol) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching diclofenac and misoprostol to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. CW121P

Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising diclofenac and misoprostol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and diclofenac and misoprostol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Diclofenac and misoprostol preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be CW121P

conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting diclofenac and misoprostol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering diclofenac and misoprostol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, diclofenac and misoprostol are released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, diclofenac and misoprostol are released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and diclofenac and misoprostol are released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, diclofenac and misoprostol are released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, diclofenac and misoprostol are released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the poljφeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching diclofenac and misoprostol to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and CW121P

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, diclofenac and misoprostol and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize diclofenac and misoprostol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of diclofenac and misoprostol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action. CW121P

The composition of the invention comprises diclofenac and misoprostol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the poljφeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular CW121P

bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. T φically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stabihty of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment. CW121P

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

10 CW121P

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the

1 1 CW121P

prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be

12 CW121P

attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, diclofenac and misoprostol are covalently attached to the polypeptide via the carboxylic acid and alcohol groups, respectively.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier poljφeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own

13 CW121P

associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-diclofenac and misoprostol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours

14 CW121P

at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terrninus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for

15 CW121P

several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW121P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and diclofenac and misoprostol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein diclofenac and misoprostol are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW121P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein diclofenac and misoprostol are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing diclofenac and misoprostol from said composition in a pH-dependent manner.

19. A method for protecting diclofenac and misoprostol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of diclofenac and misoprostol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching diclofenac and misoprostol to said polypeptide.

21. A method for delivering diclofenac and misoprostol to a patient comprising administering to said patient a composition comprising: a polypeptide; and diclofenac and misoprostol covalently attached to said polypeptide.

22. The method of claim 21 wherein diclofenac and misoprostol are released from said composition by an enzyme-catalyzed release.

18 CW121P

23. The method of claim 21 wherein diclofenac and misoprostol are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and diclofenac and misoprostol covalently attached to the polypeptide. Also provided is a method for delivery- of diclofenac and misoprostol to a patient comprising administering to the patient a composition comprising a polypeptide and diclofenac and misoprostol covalently attached to the polypeptide. Also provided is a method for protecting diclofenac and misoprostol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of diclofenac and misoprostol from a composition comprising covalently attaching it to the polypeptide.

19 CW122P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DICYCLOMINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to dicyclomine, as well as methods for protecting and administering dicyclomine. This novel compound, refeoed to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Dicyclomine is a known pharmaceutical agent that is-used in the treatment of functional disturbances of GI motility such as irritable bowel syndrome. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW122P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW122P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydrox φropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrol rtic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in thp case of naproxen-linked dextran, which is digested almost exclusively in CW122P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (dicyclomine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching dicyclomine to the N-terminus, the C-teoninus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising dicyclomine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and dicyclomine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Dicyclomine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the poljφeptide. In a prefened embodiment, the active agent is a CW122P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting dicyclomine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering dicyclomine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, dicyclomine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, dicyclomine is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and dicyclomine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, dicyclomine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, dicyclomine is released from the CW122P

composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching dicyclomine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, dicyclomine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW122P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize dicyclomine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of dicyclomine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises dicyclomine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and pol φeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary structure and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW122P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW122P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW122P

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drug-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW122P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or poljφeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-teoninus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW122P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW122P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-dicyclomine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW122P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW122P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW122P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and dicyclomine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein dicyclomine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW122P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein dicyclomine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing dicyclomine from said composition in a pH-dependent manner.

19. A method for protecting dicyclomine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of dicyclomine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching dicyclomine to said polypeptide.

21. A method for delivering dicyclomine to a patient comprising administering to said patient a composition comprising: a polypeptide; and dicyclomine covalently attached to said polypeptide.

22. The method of claim 21 wherein dicyclomine is released from said composition by an enzyme-catalyzed release.

17 CW122P

23. The method of claim 21 wherein dicyclomine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW122P

Abstract

A composition comprising a polypeptide and dicyclomine covalently attached to the polypeptide. Also provided is a method for delivery of dicyclomine to a patient comprising administering to the patient a composition comprising a polypeptide and dicyclomine covalently attached to the polypeptide. Also provided is a method for protecting dicyclomine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of dicyclomine from a composition comprising covalently attaching it to the polypeptide.

19 CW123P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DIDANOSINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to didanosine, as well as methods for protecting and administering didanosine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the, pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Didanosine is a known phaonaceutical agent that is used in the treatment of HIV. Its chemical name is 2',3'-dideoxyinosine. Its structure is:

o

^°xx)

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW123P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW123P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release. t

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via. a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW123P

Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(didanosine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching didanosine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the poljφeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising didanosine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and didanosine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Didanosine preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW123P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting didanosine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering didanosine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, didanosine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, didanosine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and didanosine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, didanosine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, didanosine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the CW123P

composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching didanosine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with, a second agent, didanosine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. ²³⁶¹ CW123P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize didanosine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of didanosine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Didanosine is the subject of U.S. Patent Numbers 4,861,759 and 5,616,566, herein incoφorated by reference, which describes how to make that drug.

# The composition of the invention comprises didanosine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW123P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW123P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW123P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ • 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant CW123P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydrox φropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW123P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, didanosine is covalently attached to the polypeptide via the ribose alcohol group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW123P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-didanosine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW123P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0 C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW123P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW123P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and didanosine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein didanosine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 CW123P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein didanosine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing didanosine from said composition in a pH-dependent manner.

19. A method for protecting didanosine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of didanosine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching didanosine to said polypeptide.

21. A method for delivering didanosine to a patient comprising administering to said patient a composition comprising: a polypeptide; and didanosine covalently attached to said polypeptide.

22. The method of claim 21 wherein didanosine is released from said composition by an enzyme-catalyzed release.

17 CW123P

23. The method of claim 21 wherein didanosine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW123P

Abstract

A composition comprising a polypeptide and didanosine covalently attached to the polypeptide. Also provided is a method for delivery of didanosine to a patient comprising administering to the patient a composition comprising a polypeptide and didanosine covalently attached to the polypeptide. Also provided is a method for protecting didanosine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of didanosine from a composition comprising covalently attaching it to the polypeptide.

19 CW124P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DIGOXIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to digoxin, as well as methods for protecting and administering digoxin. This novel compound, refeoed to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Digoxin is a known pharmaceutical agent that is used in the treatment of digitalization and maintenance therapy. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

CW124P

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW124P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW124P

linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral.adminisfration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (digoxin) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching digoxin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or poljφeptide, also refeoed to herein as a carrier peptide. In certain applications, the poljφeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising digoxin microencapsulated by a poljφeptide.

The invention provides a composition comprising a polypeptide and digoxin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, CW124P

(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Digoxin preferably is covalently attached to a side chain, the N-terminus or the C- terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the a juvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting digoxin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering digoxin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the CW124P

polypeptide. In a prefeoed embodiment, digoxin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, digoxin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and digoxin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, digoxin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, digoxin is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the poljφeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching digoxin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, digoxin and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a CW124P

carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize digoxin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of digoxin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises digoxin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and poljφeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure. CW124P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW124P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW124P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) ^' 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 ²³⁸⁴ CW124P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW124P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, poljrtyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, digoxin is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixmres of amino acid N-carboxyanhydrides.

12 CW124P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-digoxin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW124P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C- erminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW124P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptarie to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW124P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW124P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and digoxin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropol er of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein digoxin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW124P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein digoxin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said poljφeptide is capable of releasing digoxin from said composition in a pH-dependent manner.

19. A method for protecting digoxin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of digoxin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching digoxin to said polypeptide.

21. A method for delivering digoxin to a patient comprising administering to said patient a composition comprising: a polypeptide; and digoxin covalently attached to said polypeptide.

22. The method of claim 21 wherein digoxin is released from said composition by an enzyme-catalyzed release. CW124P

23. The method of claim 21 wherein digoxin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and digoxin covalently attached to the polypeptide. Also provided is a method for delivery of digoxin to a patient comprising administering to the patient a composition comprising a polypeptide and digoxin covalently attached to the polypeptide. Also provided is a method for protecting digoxin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of digoxin from a composition comprising covalently attaching it to the polypeptide.

19 ²³⁹³ CW125P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DILTIAZEM AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to diltiazem, as well as methods for protecting and administering diltiazem. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Diltiazem is a known pharmaceutical agent that is used in the tteatment of hypertension and angina. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW125P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several ²³⁹⁵ CW125P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW125P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexfran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (diltiazem) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching diltiazem to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising diltiazem microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and diltiazem covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids. CW125P

Diltiazem preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting diltiazem from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering diltiazem to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, diltiazem is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, diltiazem is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and diltiazem is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, diltiazem is released ²³⁹⁸ CW125P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, diltiazem is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching diltiazem to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, diltiazem and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. ²³⁹⁹ CW125P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize diltiazem and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of diltiazem. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Diltiazem is the subject of U.S. Patent Number 5,529,791, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises diltiazem covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopoljmier of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW125P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW125P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW125P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 1 13 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW125P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N-

11 CW125P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides.

12 CW125P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane fransport system is intrinsically asj nmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catal_/tic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-diltiazem conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW125P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AciάVN-termύius conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW125P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributyl amine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent:

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring.it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW125P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW125P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and diltiazem covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said poljφeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said poljφeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropol er of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein diltiazem is covalently attached to a side chain, the N-teoninus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW125P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein diltiazem is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing diltiazem from said composition in a pH-dependent manner.

19. A method for protecting diltiazem from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of diltiazem from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching diltiazem to said polypeptide.

21. A method for delivering diltiazem to a patient comprising administering to said patient a composition comprising: a polypeptide; and diltiazem covalently attached to said polypeptide.

22. The method of claim 21 wherein diltiazem is released from said composition by an enzyme-catalyzed release. CW125P

23. The method of claim 21 wherein diltiazem is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and diltiazem covalently attached to the polypeptide. Also provided is a method for delivery of diltiazem to a patient comprising administering to the patient a composition comprising a polypeptide and diltiazem covalently attached to the polypeptide. Also provided is a method for protecting diltiazem from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of diltiazem from a composition comprising covalently attaching it to the polypeptide.

19 CW126P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DIPYRIDAMOLE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to dipyridamole, as well as methods for protecting and administering dipyridamole. This novel compound, refened to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Dipyridamole is a known pharmaceutical agent that is used as an adjunct to coumarin anticoagulants in the prevention of postoperative thromboembolic complications of cardiac valve replacement. It is both commercially available and readily manufacmred using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered CW126P

product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations. CW126P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. -Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW126P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (dipyridamole) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching dipyridamole to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising dipyridamole microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and dipyridamole covalently attached to the poljφeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW126P

heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Dipyridamole preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting dipyridamole from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering dipyridamole to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, dipyridamole is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, dipyridamole is released in a time-dependent manner based on the pharmacokinetics of the enzyme- CW126P

catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and dipyridamole is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, dipyridamole is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, dipyridamole is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching dipyridamole to a side chain of an amino acid to form an active agent amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, dipyridamole and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the CW126P

glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize dipyridamole and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of dipyridamole. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises dipyridamole covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopoljmier of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary structure and the spatial anangement of the side chains constitute the tertiary structure. ²⁴¹⁹ CW126P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW126P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW126P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 1 13 Aspirin 180

Isoleucine 1 13 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW126P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-tenninus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide canier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 ²⁴²³ CW126P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, dipyridamole is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixmres of amino acid N-carboxyanhydrides.

12 CW126P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre- first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asj unetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-dipyridamole conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW126P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW126P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW126P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW126P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and dipyridamole covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein dipyridamole is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW126P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein dipyridamole is conformatiorially protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing dipyridamole from said composition in a pH-dependent manner.

19. A method for protecting dipyridamole from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of dipyridamole from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching dipyridamole to said polypeptide.

21. A method for delivering dipyridamole to a patient comprising administering to said patient a composition comprising: a polypeptide; and dipyridamole covalently attached to said polypeptide.

22. The method of claim 21 wherein dipyridamole is released from said composition by an enzyme-catalyzed release.

18 CW126P

23. The method of claim 21 wherein dipyridamole is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and dipyridamole covalently attached to the polypeptide. Also provided is a method for delivery of dipyridamole to a patient comprising administering to the patient a composition comprising a polypeptide and dipyridamole covalently attached to the polypeptide. Also provided is a method for protecting dipyridamole from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of dipyridamole from a composition comprising covalently attaching it to the polypeptide.

19 CW127P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DIVALPROEX AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to divalproex, as well as methods for protecting and administering divalproex. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Divalproex is a known pharmaceutical agent that is used in the tteatment of epilepsy, migraine, schizophrenia and depression. Its chemical name is 2- propylpentanoic acid. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken CW127P

under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent CW127P

in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR CW127P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (divalproex) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching divalproex to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational p'rotection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising divalproex microencapsulated by a poljφeptide.

The invention provides a composition comprising a polypeptide and divalproex covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Divalproex preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is CW127P

an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting divalproex from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering divalproex to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, divalproex is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, divalproex is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and divalproex is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, divalproex is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, divalproex is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the CW127P

composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching divalproex to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with, a second agent, divalproex and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW127P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize divalproex and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of divalproex. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Divalproex is the subject of U.S. Patent Numbers 4,988,731 and 5,212,326, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises divalproex covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW127P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. ^{2 39} CW127P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW127P

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW127P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW127P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, divalproex is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW127P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-divalproex conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW127P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimefhylaminopyridine, triethylamine or tributylamine.

14 CW127P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW127P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and divalproex covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein divalproex is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW127P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein divalproex is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing divalproex from said composition in a pH-dependent manner.

19. A method for protecting divalproex from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of divalproex from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching divalproex to said polypeptide.

21. A method for delivering divalproex to a patient comprising administering to said patient a composition comprising: a polypeptide; and divalproex covalently attached to said polypeptide.

22. The method of claim 21 wherein divalproex is released from said composition by an enzyme-catalyzed release.

17 CW127P

23. The method of claim 21 wherein divalproex is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 ²⁴⁴⁹ CW127P

Abstract

A composition comprising a polypeptide and divalproex covalently attached to the polypeptide. Also provided is a method for delivery of divalproex to a patient comprising administering to the patient a composition comprising a polypeptide and divalproex covalently attached to the polypeptide. Also provided is a method for protecting divalproex from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of divalproex from a composition comprising covalently attaching it to the polypeptide.

19 CW128P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING D-METHYLPHENIDATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to D-methylphenidate, as well as methods for protecting and administering D-methylphenidate. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

D-methylphenidate is a known pharmaceutical agent that is used in the treatment of attention deficit disorder. Its chemical name is (alphaR,2R)-alpha-phenyl-2- piperidineacetic acid methyl ester. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW128P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent,,such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW128P

reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW128P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (D- methylphenidate) to a pol ier of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching D- methylphenidate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising D-methylphenidate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and D- methylphenidate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

D-methylphenidate preferably is covalently attached to a side chain, the N- terminus or the C-teoninus of the polypeptide. In a prefeoed embodiment, the active ²⁴⁵⁴ CW128P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting D-methylphenidate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering D-methylphenidate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, D-methylphenidate is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, D-methylphenidate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and D-methylphenidate is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, D-methylphenidate is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefened embodiment, D- ²⁴⁵⁵ CW128P

methylphenidate is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching D-methylphenidate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, D-methylphenidate and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW128P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize D-methylphenidate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of D- methylphenidate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

D-methylphenidate is the subject of U.S. Patent Number 2,507,631 (1950) and

WO 99/16439 (1999), based on US application Number 937684 (1997), each of which is herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises D-methylphenidate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW128P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the'overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW128P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW128P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 1 13 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 ²⁴⁶⁰ CW128P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW128P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, D-methylphenidate is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW128P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asjαnmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active-agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-D-methylphenidate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW128P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triefhylamine or tributylamine.

14 CW128P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW128P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and D-methylphenidate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein D-methylphenidate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 CW128P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein D-methylphenidate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing D-methylphenidate from said composition in a pH-dependent manner.

19. A method for protecting D-methylphenidate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of D-methylphenidate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching D-methylphenidate to said polypeptide.

21. A method for delivering D-methylphenidate to a patient comprising administering to said patient a composition comprising: a polypeptide; and D-methylphenidate covalently attached to said polypeptide.

22. The method of claim 21 wherein D-methylphenidate is released from said composition by an enzyme-catalyzed release.

17 CW128P

23. The method of claim 21 wherein D-methylphenidate is released from said composition by a pH -dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW128P

Abstract

A composition comprising a polypeptide and D-methylphenidate covalently attached to the polypeptide. Also provided is a method for delivery of D- methylphenidate to a patient comprising administering to the patient a composition comprising a polypeptide and D-methylphenidate covalently attached to the polypeptide. Also provided is a method for protecting D-methylphenidate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for confrolling release of D-methylphenidate from a composition comprising covalently attaching it to the polypeptide.

19 CW129P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING

DOLASETROM MESYLATE AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to dolasefrόm mesylate, as well as methods for protecting and administering dolasetrom mesylate. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Dolasetrom mesylate is a known phaimaceutical agent that is used in the treatment of nausea and vomiting associated with chemotherapy. Its chemical name is 1 H-indole-3-carboxylic acid trans-octahydro-3-oxo-2,6-methano-2H-quinolizin-8-yl ester. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW129P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW129P

reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW129P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (dolasetrom mesylate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching dolasetrom mesylate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or poljφeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising dolasetrom mesylate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and dolasetrom mesylate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Dolasetrom mesylate preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active ²⁴⁷³ CW129P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. ^' In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting dolasetrom mesylate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering dolasetrom mesylate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, dolasetrom mesylate is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, dolasetrom mesylate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and dolasetrom mesylate is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, dolasetrom mesylate is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, dolasetrom CW129P

mesylate is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching dolasetrom mesylate to a side chain of an amino acid to form an active agent amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, dolasetrom mesylate and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW129P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize dolasetrom mesylate and prevent its- digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of dolasetrom mesylate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Dolasetrom mesylate is the subject of U.S. Patent Number 4,906,775, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises dolasetrom mesylate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW129P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW129P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide. •

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW129P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 1 13 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW129P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW129P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, poljrtyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW129P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-dolasetrom mesylate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW129P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triefhylamine or tributylamine.

14 CW129P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 ²⁴⁸⁴ CW129P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and dolasetrom mesylate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein dolasetrom mesylate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 ²⁴⁸⁵ CW129P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein dolasetrom mesylate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing dolasetrom mesylate from said composition in a pH-dependent manner.

19. A method for protecting dolasetrom mesylate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of dolasetrom mesylate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching dolasetrom mesylate to said poljφeptide.

21. A method for delivering dolasetrom mesylate to a patient comprising administering to said patient a composition comprising: a polypeptide; and dolasetrom mesylate covalently attached to said polypeptide.

22. The method of claim 21 wherein dolasetrom mesylate is released from said composition by an enzyme-catalyzed release.

17 CW129P

23. The method of claim 21 wherein dolasefrom mesylate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said poljφeptide.

CW129P

Abstract

A composition comprising a polypeptide and dolasefrom mesylate covalently attached to the polypeptide. Also provided is a method for delivery of dolasetrom mesylate to a patient comprising administering to the patient a composition comprising a poljφeptide and dolasetrom mesylate covalently attached to the polypeptide. Also provided is a method for protecting dolasefrom mesylate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of dolasefrom mesylate from a composition comprising covalently attaching it to the polypeptide.

19 CW130P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DONEPEZIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to donepezil, as well as methods for protecting and administering donepezil. This novel compound, refened to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaimaceutical effectiveness.

BACKGROUND OF THE INVENTION

Donepezil is a known pharmaceutical agent that is used in the treatment of Alzheimer's and attention deficit disorder. Its chemical name is 2,3-dihydro-5,6- dimethoxy-2-[[l-(phenylmethyl)-4-piperidinyl]methyl]-l H-inden-1-one. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW130P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach m « ilieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW130P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW130P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (donepezil) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching donepezil to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Altematively, the present invention provides a pharmaceutical composition comprising donepezil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and donepezil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Donepezil preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW130P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- te ninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting donepezil from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering donepezil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, donepezil is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, donepezil is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and donepezil is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, donepezil is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, donepezil is released from the composition in a sustained release. ²⁴⁹³ CW130P

In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching donepezil to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, donepezil and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is CW130P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize donepezil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of donepezil. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Donepezil is the subject of U.S. Patent Number 4,895,841 , herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises donepezil covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW130P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's ²⁴⁹⁶ CW130P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW130P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 1 13 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW130P

delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW130P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW130P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-donepezil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW130P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terrninus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimefhylaminopyridine, triefhylamine or tributylamine.

14 CW130P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl GIutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 ²⁵⁰³ CW130P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and donepezil covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein donepezil is covalently attached to a side chain, the N-teoninus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 CW130P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein donepezil is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing donepezil from said composition in a pH-dependent manner.

19. A method for protecting donepezil from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of donepezil from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching donepezil to said polypeptide.

21. A method for delivering donepezil to a patient comprising administering to said patient a composition comprising: a polypeptide; and donepezil covalently attached to said polypeptide.

22. The method of claim 21 wherein donepezil is released from said composition by an enzyme-catalyzed release.

17 CW130P

23. The method of claim 21 wherein donepezil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 2506 „-- „ „,,-,

CW130P

Abstract

A composition comprising a polypeptide and donepezil covalently attached to the polypeptide. Also provided is a method for delivery of donepezil to a patient comprising administering to the patient a composition comprising a polypeptide and donepezil covalently attached to the polypeptide. Also provided is a method for protecting donepezil from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of donepezil from a composition comprising covalently attaching it to the polypeptide.

19 ²⁵⁰⁷ CW131P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DOPAMINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to dopamine, as well as methods for protecting and administering dopamine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Dopamine is a known pharmaceutical agent that is used used to increase cardiac output, blood pressure, and urine flow as an adjunct in the treatment of shock that persists after adequate fluid volume replacement. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW131P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage ²⁵⁰⁹ CW131P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW131P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (dopamine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching dopamine to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising dopamine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and dopamine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Dopamine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW131P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting dopamine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering dopamine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, dopamine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, dopamine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and dopamine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, dopamine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, dopamine is released from the composition in a sustained release. CW131P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching dopamine to a side chain of an amino acid to form an active agent/amino acid complex; (b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, dopamine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW131P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize dopamine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of dopamine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises dopamine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW131P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW131P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. ^' As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW131P

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW131P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW131P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, dopamine is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW131P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-dopamine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW131P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-ter minus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 ²⁵²¹ CW131P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AIkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW131P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and dopamine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said poljφeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein dopamine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 CW131P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein dopamine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing dopamine from said composition in a pH-dependent manner.

19. A method for protecting dopamine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of dopamine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching dopamine to said polypeptide.

21. A method for delivering dopamine to a patient comprising administering to said patient a composition comprising: a polypeptide; and dopamine covalently attached to said polypeptide.

22. The method of claim 21 wherein dopamine is released from said composition by an enzyme-catalyzed release.

17 CW131P

23. The method of claim 21 wherein dopamine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said poljφeptide.

CW131P

Abstract

A composition comprising a polypeptide and dopamine covalently attached to the polypeptide. Also provided is a method for delivery of dopamine to a patient comprising administering to the patient a composition comprising a polypeptide and dopamine covalently attached to the polypeptide. Also provided is a method for protecting dopamine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of dopamine from a composition comprising covalently attaching it to the polypeptide.

19 ²⁵²⁶ CW132P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DOXAZOSIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to doxazosin, as well as methods for protecting and administering doxazosin. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Doxazosin is a known phaonaceutical agent that is used in the treatment of hypertension. Its chemical name is l-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-[(2,3- dihydro-l,4-benzodioxin-2-yl)carbonyl]piperizine. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW132P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW132P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in tum, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW132P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (doxazosin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching doxazosin to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising doxazosin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and doxazosin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Doxazosin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW132P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting doxazosin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering doxazosin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, doxazosin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, doxazosin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and doxazosin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, doxazosin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, doxazosin is released from the composition in a sustained release. CW132P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching doxazosin to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, doxazosin and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW132P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize doxazosin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of doxazosin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Doxazosin is the subject of U.S. Patent Number GB 2007656 B (1982), herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises doxazosin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW132P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW132P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW132P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 1 13 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW132P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide canier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW132P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzjmies in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, doxazosin is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW132P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-doxazosin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW132P

AcidTN- terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Arnine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The cmde product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW132P

Preparation of γ-AIkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AIkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 ²⁵⁴¹ CW132P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and doxazosin covalently attached to said poljφeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein doxazosin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 ²⁵⁴² CW132P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein doxazosin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing doxazosin from said composition in a pH-dependent manner.

19. A method for protecting doxazosin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of doxazosin from a composition wherein said composition comprises a poljφeptide, said method comprising covalently attaching doxazosin to said polypeptide.

21. A method for delivering doxazosin to a patient comprising administering to said patient a composition comprising: a polypeptide; and doxazosin covalently attached to said polypeptide.

22. The method of claim 21 wherein doxazosin is released from said composition by an enzyme-catalyzed release.

17 CW132P

23. The method of claim 21 wherein doxazosin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW132P

Abstract

A composition comprising a polypeptide and doxazosin covalently attached to the polypeptide. Also provided is a method for delivery of doxazosin to a patient comprising administering to the patient a composition comprising a polypeptide and doxazosin covalently attached to the polypeptide. Also provided is a method for protecting doxazosin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of doxazosin from a composition comprising covalently attaching it to the polypeptide.

19 CW133P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DOXORUBICIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to doxorabicin, as well as methods for protecting and administering doxorabicin. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaimaceutical effectiveness.

BACKGROUND OF THE INVENTION

Doxorabicin is a known pharmaceutical agent that is used in the treatment of bacterial infection. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW133P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent,, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW133P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of poly aspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW133P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (doxorabicin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching doxorabicin to the N-terminus,' the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising doxorabicin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and doxorabicin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Doxorabicin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW133P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting doxorabicin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering doxorabicin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, doxorabicin is released from the composition • by an enzyme-catalyzed release. In another prefeoed embodiment, doxorabicin is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and doxorabicin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, doxorabicin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, doxorabicin is released from the CW133P

composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching doxorubicin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, doxorabicin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW133P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize doxorabicin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of doxorabicin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Doxorabicin is the subject of U.S. Patent Number 4,837,028, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises doxorabicin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW133P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW133P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW133P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and s #o active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW133P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-dmg moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW133P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these poljφeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, doxorabicin is covalently attached to the polypeptide via the alcohol group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW133P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active -agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-doxorabicin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW133P

Acid/N-ter minus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triefhylamine or tributylamine.

14 CW133P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW133P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and doxorabicin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein doxorabicin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW133P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein doxorabicin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing doxorabicin from said composition in a pH-dependent manner.

19. A method for protecting doxorabicin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of doxorabicin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching doxorabicin to said polypeptide.

21. A method for delivering doxorabicin to a patient comprising administering to said patient a composition comprising: a polypeptide; and doxorabicin covalently attached to said polypeptide.

22. The method of claim 21 wherein doxorabicin is released from said composition by an enzyme-catalyzed release.

17 ²⁵⁶² CW133P

23. The method of claim 21 wherein doxorabicin is released from said composition by a pH -dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW133P

Abstract

A composition comprising a polypeptide and doxorabicin covalently attached to the polypeptide. Also provided is a method for delivery of doxorabicin to a patient comprising administering to the patient a composition comprising a polypeptide and doxorabicin covalently attached to the polypeptide. Also provided is a method for protecting doxorabicin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of doxorabicin from a composition comprising covalently attaching it to the polypeptide.

19 CW134P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DULOXETINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to duloxetine, as well as methods for protecting and administering duloxetine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Duloxetine is a known pharmaceutical agent that is used in the treatment of depression. Its chemical name is (S)-N-methyl-gamma-(l-naphthalenyloxy)-2- thiophenepropanamine. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW134P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW134P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW134P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (duloxetine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching duloxetine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising duloxetine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and duloxetine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Duloxetine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW134P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting duloxetine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering duloxetine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, duloxetine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, duloxetine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and duloxetine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, duloxetine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, duloxetine is released from the composition in a sustained release. CW134P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching duloxetine to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, duloxetine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW134P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize duloxetine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of duloxetine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Duloxetine is the subject of 273658 B (1990), priority US 945122 (1986), herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises duloxetine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW134P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW134P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW134P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW134P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-dmg dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW134P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, pol yrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, duloxetine is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW134P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catal ftic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-duloxetine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

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Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimefhylaminopyridine, triethylamine or tributylamine.

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Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW134P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and duloxetine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopoljmier of a namrally occurring amino acid.

_*

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopoljmier of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein duloxetine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein duloxetine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing duloxetine from said composition in a pH-dependent manner.

19. A method for protecting duloxetine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of duloxetine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching duloxetine to said polypeptide.

21. A method for delivering duloxetine to a patient comprising administering to said patient a composition comprising: a polypeptide; and duloxetine covalently attached to said polypeptide.

22. The method of claim 21 wherein duloxetine is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein duloxetine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

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Abstract

A composition comprising a polypeptide and duloxetine covalently attached to the polypeptide. Also provided is a method for delivery of duloxetine to a patient comprising administering to the patient a composition comprising a polypeptide and duloxetine covalently attached to the polypeptide. Also provided is a method for protecting duloxetine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of duloxetine from a composition comprising covalently attaching it to the polypeptide.

19 CW135P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING DUTASTERIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to dutasteride, as well as methods for protecting and administering dutasteride. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Dutasteride is a known pharmaceutical agent that is used in the treatment of benign prostate hypertrophy and alopecia. Its chemical name is (4aR,4bS,6aS,7S,9aS, 9bS,l laR)-N-[2,5-bis(trifluoromethyl)phenyl]-2,4a,4b,5,6,6a,7,8,9,9a,9b,10,l 1,1 la- tetradecahydro-4a,6a-dimethyl-2-oxo-lH- indeno[5,4-f]quinoline-7-carboxamide. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW135P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW135P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW135P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (dutasteride) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching dutasteride to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising dutasteride microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and dutasteride covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids. CW135P

Dutasteride preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting dutasteride from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering dutasteride to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, dutasteride is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, dutasteride is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and dutasteride is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, dutasteride is CW135P

released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, dutasteride is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching dutasteride to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, dutasteride and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW135P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize dutasteride and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of dutasteride. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Dutasteride is the subject of WO 95/7927 (1995), priority US 123280 (1993), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises dutasteride covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure. CW135P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW135P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW135P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW135P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-dmg conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW135P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, dutasteride is covalently attached to the polypeptide via the amino groups.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixmres of amino acid N-carboxyanhydrides.

12 CW135P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre- first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane fransport system is intrinsically as imetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-dutasteride conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW135P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW135P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl Glutanτate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW135P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW135P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and dutasteride covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropoljmier of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein dutasteride is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

17 CW135P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein dutasteride is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing dutasteride from said composition in a pH-dependent manner.

19. A method for protecting dutasteride from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of dutasteride from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching dutasteride to said polypeptide.

21. A method for delivering dutasteride to a patient comprising administering to said patient a composition comprising: , a polypeptide; and dutasteride covalently attached to said polypeptide.

22. The method of claim 21 wherein dutasteride is released from said composition by an enzyme-catalyzed release.

18 CW135P

23. The method of claim 21 wherein dutasteride is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and dutasteride covalently attached to the polypeptide. Also provided is a method for delivery of dutasteride to a patient comprising administering to the patient a composition comprising a polypeptide and dutasteride covalently attached to the polypeptide. Also provided is a method for protecting dutasteride from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of dutasteride from a composition comprising covalently attaching it to the polypeptide.

19 CW136P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ECADOTRIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ecadottil, as well as methods for protecting and administering ecadottil. This novel compound, refened to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ecadottil is a known pharmaceutical agent that is used in the tteatment of hypertension, heart failure and ciohosis. Its chemical name is (S)-N-[2-

[(acetylthio)methyl]-l-oxo-3-phenylpropyl]glycine phenyl methyl ester. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW136P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW136P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW136P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ecadotril) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching ecadotril to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ecadotril microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ecadotril covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Ecadotril preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW136P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ecadotril from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ecadotril to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, ecadotril is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, ecadotril is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ecadotril is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, ecadotril is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ecadotril is released from the composition in a sustained release. CW136P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ecadotril to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ecadotril and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW136P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ecadotril and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ecadotril. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Ecadotril is the subject of EP 318377 B (1993), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises ecadotril covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW136P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW136P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW136P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 1 13 Aspirin 180

Isoleucine 1 13 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW136P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide canier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-dmg moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW136P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these poljφeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ecadotril is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW136P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ecadotril conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW136P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimefhylaminopyridine, triefhylamine or tributylamine.

14 CW136P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW136P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ecadotril covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said poljφeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ecadotril is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 CW136P

12. The composition of claim 1 1 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ecadotril is conformationally protected by folding of said poljφeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ecadotril from said composition in a pH-dependent manner.

19. A method for protecting ecadotril from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ecadotril from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ecadotril to said polypeptide.

21. A method for delivering ecadotril to a patient comprising administering to said patient a composition comprising: a polypeptide; and ecadotril covalently attached to said polypeptide.

22. The method of claim 21 wherein ecadotril is released from said composition by an enzyme-catalyzed release.

17 CW136P

23. The method of claim 21 wherein ecadotril is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW136P

Abstract

A composition comprising a polypeptide and ecadotril covalently attached to the polypeptide. Also provided is a method for delivery of ecadotril to a patient comprising administering to the patient a composition comprising a polypeptide and ecadotril covalently attached to the polypeptide. Also provided is a method for protecting ecadotril from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for confrolling release of ecadotril from a composition comprising covalently attaching it to the polypeptide.

19 CW137P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ECOPIPAM AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ecopipam, as well as methods for protecting and administering ecopipam. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ecopipam is a known pharmaceutical agent that is used in the treatment of obesity. Its chemical name is (6aS,13bR)-l l-chloro-6,6a,7,8,9,13b-hexahydro-7-methyl- 5H-benzo[d ]naphth[2,l-b]azepin-12-ol. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW137P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW137P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW137P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ecopipam) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ecopipam to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ecopipam microencapsulated by a poljφeptide.

The invention provides a composition comprising a polypeptide and ecopipam covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ecopipam preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW137P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is. an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ecopipam from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ecopipam to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, ecopipam is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, ecopipam is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ecopipam is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, ecopipam is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ecopipam is released from the composition in a sustained release. CW137P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ecopipam to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ecopipam and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW137P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ecopipam and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ecopipam. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Ecopipam is the subject of EP 254737 A (1990), priority U.S. application 820471

(1986), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises ecopipam covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW137P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW137P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW137P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 1 13 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW137P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW137P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Altematively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ecopipam is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW137P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ecopipam conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW137P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptjde carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

AIcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triefhylamine or tributylamine.

14 CW137P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0 C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 ²⁶³⁶ CW137P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ecopipam covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ecopipam is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 CW137P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ecopipam is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ecopipam from said composition in a pH-dependent manner.

19. A method for protecting ecopipam from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ecopipam from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ecopipam to said polypeptide.

21. A method for delivering ecopipam to a patient comprising administering to said patient a composition comprising: a polypeptide; and ecopipam covalently attached to said polypeptide.

22. The method of claim 21 wherein ecopipam is released from said composition by an enzyme-catalyzed release.

17 CW137P

23. The method of claim 21 wherein ecopipam is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW137P

Abstract

A composition comprising a polypeptide and ecopipam covalently attached to the polypeptide. Also provided is a method for delivery of ecopipam to a patient comprising administering to the patient a composition comprising a polypeptide and ecopipam covalently attached to the polypeptide. Also provided is a method for protecting ecopipam from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ecopipam from a composition comprising covalently attaching it to the polypeptide.

19 CW138P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING EDODEKIN ALFA AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to edodekin alfa (interleukin-12), as well as methods for protecting and administering edodekin alfa. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Interleukin-12 is a heterodimeric cytokine produced by phagocytic cells, professional antigen-presenting cells such as dendritic cells and skin Langerhans cells, and B cells. Interleukin-12 production is induced by bacteria, intracellular pathogens, fungi, viruses, or their products in a T-cell-independent pathway or a T-cell-dependent pathway, the latter mediated through CD40 ligand-CD40 interaction. Interleukin-12 is produced rapidly after infection and acts as a proinflammatory cytokine eliciting production of interferon gamma, by T and natural killer cells, which activates phagocytic cells. The production of interleukin-12 is strictly regulated by positive and negative feedback mechanisms. If interleukin-12 and interleukin-12-induced interferon gamma are present during early T-cell expansion in response to antigen, T-helper type-1 cell generation is favored and generation of T-helper type-2 cells is inhibited. Thus interleukin-12 is also a potent immunoregulatory cytokine that promotes T-helper type-1 differentiation and is instrumental in the T-helper type-1 -dependent resistance to infections by bacteria, intracellular parasites, fungi, and certain viruses. By inhibiting T- helper type-2 cell response, interleukin-12 has a suppressive effect on allergic reactions; by promoting T-helper type-1 responses it participates in the immunopathology responsible for several organ-specific autoimmune diseases. Viruses inducing a CW138P

permanent or transient immunodepression, such as HIV and measles, may act, in part, by suppressing interleukin-12 production. Because of its ability to enhance resistance to several infectious diseases and to act as an adjuvant in vaccination, and because of its powerful antitumor effect in vivo, interleukin-12 is cuoently in clinical trials in cancer patients and HIV-infected patients, and it is being considered for therapeutic use in other diseases.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme CW138P

degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is umeliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide CW138P

spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in tum, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (edodekin alfa) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching edodekin alfa to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the CW138P

upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Altematively, the present invention provides a pharmaceutical composition comprising edodekin alfa microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and edodekin alfa covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Edodekin alfa preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- teπninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In CW138P

another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting edodekin alfa from degradation comprising covalently attaching it to a poljφeptide.

The invention also provides a method for delivering edodekin alfa to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, edodekin alfa is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, edodekin alfa is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and edodekin alfa is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, edodekin alfa is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, edodekin alfa is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching edodekin alfa to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). CW138P

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, edodekin alfa and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize edodekin alfa and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of edodekin alfa. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises edodekin alfa covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a CW138P

heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of CW138P

maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of CW138P

the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW138P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

1 1 CW138P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 ²⁶⁵² CW138P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, edodekin alfa is covalently attached to the polypeptide via any free alcohol, amine or acid groups, or alternatively via an artificial linker.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier poljφeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW138P

mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-edodekin alfa conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AcidTN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

14 CW138P

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW138P

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW138P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and edodekin alfa covalently attached to said polypeptide. ^*

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopoljmier of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein edodekin alfa is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

17 CW139P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING EFAVIRENZ AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to efavirenz, as well as methods for protecting and administering efavirenz. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaimaceutical effectiveness.

BACKGROUND OF THE INVENTION

Efavirenz is a known pharmaceutical agent that is used in the tteatment of HIV infection. Its chemical name is (S)-6-chloro-4-(cyclopropylethynyl)-l,4-dihydro-4- (trifluoromethyl)-2H -3,l-benzoxazin-2-one. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these ²⁶⁵⁸ CW139P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble ²⁶⁵⁹ CW139P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of poly aspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW139P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (efavirenz) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching efavirenz to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising efavirenz microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and efavirenz covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Efavirenz preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW139P

the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting efavirenz from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering efavirenz to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, efavirenz is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, efavirenz is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and efavirenz is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, efavirenz is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, efavirenz is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW139P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching efavirenz to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with, a second agent, efavirenz and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW139P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize efavirenz and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of efavirenz. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Efavirenz is the subject of U.S. Patent Numbers 5,519,021, 5,663,169 and 5,811,423, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises efavirenz covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW139P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are

"pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW139P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW139P

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 ²⁶⁶⁷ CW139P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-dmg moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 ²⁶⁶⁸ CW139P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to imparjt specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, efavirenz is covalently attached to the polypeptide via the amino group or alternately through an artificial linker.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 ²⁶⁶⁹ CW139P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asjmimetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-efavirenz conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW139P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Anύne/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The cmde product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimefhylaminopyridine, triefhylamine or tributylamine.

14 CW139P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW139P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and efavirenz covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopoljmier of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropoljmier of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopoljmier of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropoljmier of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein efavirenz is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 ²⁶⁷³ CW139P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein efavirenz is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing efavirenz from said composition in a pH-dependent manner.

19. A method for protecting efavirenz from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of efavirenz from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching efavirenz to said polypeptide.

21. A method for delivering efavirenz to a patient comprising administering to said patient a composition comprising: a polypeptide; and efavirenz covalently attached to said polypeptide.

22. The method of claim 21 wherein efavirenz is released from said composition by an enzyme-catalyzed release.

17 CW139P

23. The method of claim 21 wherein efavirenz is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 ²⁶⁷⁵ CW139P

Abstract

A composition comprising a polypeptide and efavirenz covalently attached to the polypeptide. Also provided is a method for delivery of efavirenz to a patient comprising administering to the patient a composition comprising a polypeptide and efavirenz covalently attached to the polypeptide. Also provided is a method for protecting efavirenz from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for confrolling release of efavirenz from a composition comprising covalently attaching it to the polypeptide.

19 CW140P

A NOVEL PHARMACEUTICAL COMPOUND AND METHODS OF MAKING

AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to a ketolide antibiotic , as well as methods for protecting and administering a ketolide antibiotic . This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

The ketolide antibiotic of the present invention is a known pharmaceutical agent that is used in the treatment of bacterial infection. Its chemical name is

(3aS,4R,7R,9R, 1 OR, 11 R, 13R, 15R, 15aR)-4-ethyloctahydro-3a,7,9, 11,13,15-hexamethyl- 11 -[[3-(3-quinolinyl)-2-propenyl]oxy]-l 0-[[3,4,6-trideoxy-3-(dimethylamino)-beta-D- xylo-hexopyranosyl]oxy]-2H-oxacyclotefradecino[4,3-d]oxazole-2,6,8, 14( 1 H,7H,9H)- tetrone. Its stracmre is:

CW140P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release ²⁶⁷⁸ CW140P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW140P

linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (a ketolide antibiotic ) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching a ketolide antibiotic to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising a ketolide antibiotic microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and a ketolide antibiotic covalently attached to the polypeptide. Preferably, the polypeptide is (i) an ²⁶⁸⁰ CW140P

oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

A ketolide antibiotic preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting a ketolide antibiotic from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering a ketolide antibiotic to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently ²⁶⁸¹ CW140P

attached to the polypeptide. In a prefened embodiment, a ketolide antibiotic is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, a ketolide antibiotic is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and a ketolide antibiotic is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, a ketolide antibiotic is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, a ketolide antibiotic is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching a ketolide antibiotic to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, a ketolide antibiotic and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side CW140P

chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize a ketolide antibiotic and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of a ketolide antibiotic . Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

A ketolide antibiotic is the subject of WO 98/9978 (1998), priority US 707776 (1996), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises a ketolide antibiotic covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the ²⁶⁸³ CW140P

conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in. the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only ²⁶⁸⁴ CW140P

5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends pn the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection 'of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight ²⁶⁸⁵ CW140P

active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW140P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

11 ²⁶⁸⁷ CW140P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide canier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-dmg moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 ²⁶⁸⁸ CW140P

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions,

13 CW140P

sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-a ketolide antibiotic conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

14 ²⁶⁹⁰ CW140P

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents ihclude dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl GIutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 ²⁶⁹¹ CW140P

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 ²⁶⁹² CW140P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and a ketolide antibiotic covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein a ketolide antibiotic is covalently attached to a side chain, the N-terminus or the C-teoninus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

17 2693 CW140P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein a ketolide antibiotic is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing a ketolide antibiotic from said composition in a pH-dependent manner.

19. A method for protecting a ketolide antibiotic from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of a ketolide antibiotic from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching a ketolide antibiotic to said polypeptide.

21. A method for delivering a ketolide antibiotic to a patient comprising administering to said patient a composition comprising: a polypeptide; and a ketolide antibiotic covalently attached to said polypeptide.

22. The method of claim 21 wherein a ketolide antibiotic is released from said composition by an enzyme-catalyzed release.

18 ²⁶⁹⁴ CW140P

23. The method of claim 21 wherein a ketolide antibiotic is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition -further comprises an adjuvant covalently attached to said poljφeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and a ketolide antibiotic covalently attached to the polypeptide. Also provided is a method for delivery of a ketolide antibiotic to a patient comprising administering to the patient a composition comprising a polypeptide and a ketolide antibiotic covalently attached to the polypeptide. Also provided is a method for protecting a ketolide antibiotic from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of a ketolide antibiotic from a composition comprising covalently attaching it to the polypeptide.

19 CW141P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING EMIVIRINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaonaceutical compound that comprises a polypeptide that is preferably covalently attached to emivirine, as well as methods for protecting and administering emivirine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Emivirine is a known pharmaceutical agent that is used in the treatment of HIV infection. Its chemical name is l-(ethoxymethyl)-5-(l -methyl ethyl)-6-(phenylmethyl )- 2,4(lH,3H)-pyrimidinedione. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. ²⁶⁹⁶ CW141P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and fransport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage ²⁶⁹⁷ CW141P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzjmies in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in ²⁶⁹⁸ CW141P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (emivirine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching emivirine to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising emivirine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and emivirine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Emivirine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW141P

carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting emivirine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering emivirine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, emivirine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, emivirine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and emivirine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, emivirine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, emivirine is released from the composition in a sustained release. CW141P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching emivirine to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, emivirine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW141 P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize emivirine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of emivirine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Emivirine is the subject of EP 420763 B (1999), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises emivirine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropoljmier of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the stmctural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW141P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant

_* force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. T φically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's ²⁷⁰³ CW141P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW141P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 1 13 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 2705 CW141P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Altematively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW141P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-dmg dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Altematively, the active agent can be attached directly to the pendant group where some other indigenous enzjmies in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW141P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-emivirine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW141P

AcidTN-terrninus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The cmde product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 2709 CW141P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 ²7¹⁰ CW141P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and emivirine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein emivirine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 ²⁷¹¹ CW141P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein emivirine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing emivirine from said composition in a pH-dependent manner.

19. A method for protecting emivirine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of emivirine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching emivirine to said polypeptide.

21. A method for delivering emivirine to a patient comprising administering to said patient a composition comprising: a polypeptide; and emivirine covalently attached to said polypeptide.

22. The method of claim 21 wherein emivirine is released from said composition by an enzyme-catalyzed release.

17 CW141P

23. The method of claim 21 wherein emivirine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition -further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 ²⁷¹ CW141P

Abstract

A composition comprising a polypeptide and emivirine covalently attached to the polypeptide. Also provided is a method for delivery of emivirine to a patient comprising administering to the patient a composition comprising a polypeptide and emivirine covalently attached to the polypeptide. Also provided is a method for protecting emivirine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of emivirine from a composition comprising covalently attaching it to the polypeptide.

19 CW142P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ENALAPRIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to enalapril, as well as methods for protecting and administering enalapril. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Enalapril is a known pharmaceutical agent that is used in the treatment of hypertension. Its chemical name is (S)- 1 -[N-[ 1 -(ethoxycarbonyl)-3 -phenylpropyl]-L- alanyl]-L-proline. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW142P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW142P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW142P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (enalapril) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching enalapril to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the poljφeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising enalapril microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and enalapril covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Enalapril preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW142P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting enalapril from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering enalapril to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, enalapril is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, enalapril is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and enalapril is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, enalapril is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, enalapril is released from the composition in a sustained release. CW142P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching enalapril to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, enalapril and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW142P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize enalapril and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of enalapril. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Enalapril is the subject of EP 12401 (1984), priority US application 968249

(1978), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises enalapril covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW142P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW142P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW142P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW142P

delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW142P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, enalapril is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW142P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently ia specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-enalapril conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW142P

Acid/N-terrninus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Ter minus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agenf can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- . hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triefhylamine or tributylamine.

14 CW142P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW142P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and enalapril covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein enalapril is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW142P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein enalapril is conformationally protected by folding of said poljφeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing enalapril from said composition in a pH-dependent manner.

19. A method for protecting enalapril from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of enalapril from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching enalapril to said polypeptide.

21. A method for delivering enalapril to a patient comprising administering to said patient a composition comprising: a polypeptide; and enalapril covalently attached to said polypeptide.

22. The method of claim 21 wherein enalapril is released from said composition by an enzyme-catalyzed release.

17 CW142P

23. The method of claim 21 wherein enalapril is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW142P

Abstract

A composition comprising a polypeptide and enalapril covalently attached to the polypeptide. Also provided is a method for delivery of enalapril to a patient comprising administering to the patient a composition comprising a polypeptide and enalapril covalently attached to the polypeptide. Also provided is a method for protecting enalapril from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of enalapril from a composition comprising covalently attaching it to the polypeptide.

19 CW143P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ENALIPRIL

ELANAPRIL AND HYDROCHLOROTHIAZIDE AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to elanapril and hydrochlorothiazide, as well as methods for protecting and administering elanapril and hydrochlorothiazide. This novel compound, refeoed to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Elanapril and hydrochlorothiazide are known phaonaceutical agents used together in the treatment of hypertension.

The chemical name of elanapril is (S)-l-[N-[l-(ethoxycarbonyl)-3-phenylpropyl]- L-alanyl]-L-proline. Its stracture is:

The chemical stracture of hydrochlorothiazide is:

CW143P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW143P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW143P

linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (elanapril and hydrochlorothiazide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching elanapril and hydrochlorothiazide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a ■ carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising elanapril and hydrochlorothiazide microencapsulated by a polypeptide. CW143P

The invention provides a composition comprising a polypeptide and elanapril and hydrochlorothiazide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Elanapril and hydrochlorothiazide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting elanapril and hydrochlorothiazide from degradation comprising covalently attaching it to a polypeptide. CW143P

The invention also provides a method for delivering elanapril and hydrochlorothiazide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, elanapril and hydrochlorothiazide is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, elanapril and hydrochlorothiazide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and elanapril and hydrochlorothiazide is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, elanapril and hydrochlorothiazide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, elanapril and hydrochlorothiazide is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching elanapril and hydrochlorothiazide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, elanapril and hydrochlorothiazide and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the CW143P

polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize elanapril and hydrochlorothiazide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of elanapril and hydrochlorothiazide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Elanapril is the subject of of EP 12401 (1984), priority US application 968249 (1978), and U.S. patent numbers 4,374,829 and 4,472,380, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises elanapril and hydrochlorothiazide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a CW143P

sjmthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of CW143P

maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of CW143P

the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW143P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

11 CW143P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this poljmierization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW143P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and J ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, elanapril is covalently attached to the polypeptide via the carboxylic acid group; hydrochlorothiazide is attached via its amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW143P

mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-elanapril and hydrochlorothiazide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-ter minus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

14 CW143P

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW143P

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW143P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and elanapril and hydrochlorothiazide covalently attached to said poljφeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said poljφeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein elanapril and hydrochlorothiazide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW143P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the foim of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein elanapril and hydrochlorothiazide is - conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing elanapril and hydrochlorothiazide from said composition in a pH-dependent manner.

19. A method for protecting elanapril and hydrochlorothiazide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of elanapril and hydrochlorothiazide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching elanapril and hydrochlorothiazide to said polypeptide.

21. A method for delivering elanapril and hydrochlorothiazide to a patient comprising administering to said patient a composition comprising: a polypeptide; and elanapril and hydrochlorothiazide covalently attached to said polypeptide.

22. The method of claim 21 wherein elanapril and hydrochlorothiazide is released from said composition by an enzyme-catalyzed release.

18 CW143P

23. The method of claim 21 wherein elanapril and hydrochlorothiazide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and elanapril and hydrochlorothiazide covalently attached to the polypeptide. Also provided is a method for delivery of elanapril and hydrochlorothiazide to a patient comprising administering to the patient a composition comprising a polypeptide and elanapril and hydrochlorothiazide covalently attached to the polypeptide. Also provided is a method for protecting elanapril and hydrochlorothiazide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of elanapril and hydrochlorothiazide from a composition comprising covalently attaching it to the polypeptide.

19 CW144P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ENILURACIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to eniluracil, as well as methods for protecting and administering eniluracil. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Eniluracil is a known pharmaceutical agent that is used in the treatment of pancreatic and colorectal cancer. Its chemical name is 5-ethynyl-2,4(lH,3H)- pyrimidinedione. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW144P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW144P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in tum, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW144P

Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (eniluracil) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching eniluracil to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzjmiatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising eniluracil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and eniluracil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Eniluracil preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW144P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting eniluracil from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering eniluracil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, eniluracil is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, eniluracil is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and eniluracil is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, eniluracil is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, eniluracil is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW144P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching eniluracil to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, eniluracil and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW144P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize eniluracil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of eniluracil. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Eniluracil is the subject of WO 92/1452 (1992), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises eniluracil covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW144P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW144P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW144P

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoπnational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW144P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-dmg dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW144P

maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, eniluracil is covalently attached to the polypeptide via the amino groups.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW144P

invention, a pre- first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-eniluracil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW144P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/jN-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW144P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW144P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and eniluracil covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein eniluracil is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW144P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein eniluracil is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing eniluracil from said composition in a pH-dependent manner.

19. A method for protecting eniluracil from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for confrolling release of eniluracil from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching eniluracil to said polypeptide.

21. A method for delivering eniluracil to a patient comprising administering to said patient a composition comprising: a polypeptide; and eniluracil covalently attached to said polypeptide.

22. The method of claim 21 wherein eniluracil is released from said composition by an enzyme-catalyzed release.

17 CW144P

23. The method of claim 21 wherein eniluracil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW144P

Abstract

A composition comprising a polypeptide and eniluracil covalently attached to the polypeptide. Also provided is a method for delivery of eniluracil to a patient comprising administering to the patient a composition comprising a polypeptide and eniluracil covalently attached to the polypeptide. Also provided is a method for protecting eniluracil from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of eniluracil from a composition comprising covalently attaching it to the polypeptide.

19 CW145P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ENOXAPARTN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to enoxaparin, as well as methods for protecting and administering enoxaparin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaimaceutical effectiveness.

BACKGROUND OF THE INVENTION

Enoxaparin is a known pharmaceutical agent that is used in the treatment of throbosis and myocardial infarction. It is a low molecular weight heparin, and is described in U.S. Patent Numbers 4,486,420, 4,692,435, and 5,389,619, incoφorated herein by reference.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW145P

invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW145P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW145P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (enoxaparin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching enoxaparin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising enoxaparin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and enoxaparin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Enoxaparin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide. CW145P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the acti e agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting enoxaparin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering enoxaparin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, enoxaparin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, enoxaparin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and enoxaparin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, enoxaparin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, enoxaparin is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW145P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching enoxaparin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, enoxaparin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize enoxaparin and prevent its digestion in the stomach. In CW145P

addition, the pharmacologic effect can be prolonged by delayed release of enoxaparin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises enoxaparin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW145P

and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Tjφically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will CW145P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the CW145P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is - only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order

10 CW145P

to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

11 CW145P

preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, enoxaparin is covalently attached to the polypeptide via any free alcohol, amine or acid groups, or alternatively via artificial linkers.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the poljφeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

12 CW145P

intestinal fransport systems classified according to the physical properties of the fransported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-enoxaparin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to

0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

13 CW145P

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/TV-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triefhylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

14 CW145P

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW145P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and enoxaparin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein enoxaparin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW145P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein enoxaparin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing enoxaparin from said composition in a pH-dependent manner.

19. A method for protecting enoxaparin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of enoxaparin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching enoxaparin to said polypeptide.

21. A method for delivering enoxaparin to a patient comprising administering to said patient a composition comprising: a polypeptide; and enoxaparin covalently attached to said polypeptide.

22. The method of claim 21 wherein enoxaparin is released from said ' composition by an enzyme-catalyzed release.

17 CW145P

23. The method of claim 21 wherein enoxaparin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW145P

Abstract

A composition comprising a polypeptide and enoxaparin covalently attached to the polypeptide. Also provided is a method for delivery of enoxaparin to a patient comprising administering to the patient a composition comprising a polypeptide and enoxaparin covalently attached to the polypeptide. Also provided is a method for protecting enoxaparin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of enoxaparin from a composition comprising covalently attaching it to the polypeptide.

19 CW146P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING EPOETIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to epoetin, as well as methods for protecting and administering epoetin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Epoetin is a known pharmaceutical agent that is used in the tteatment of anemia. Its chemical name is 1-165-erythropoietin (human clone lambdaHEPOFL13 protein moiety) glycoform alpha.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW146P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzjmie degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW146P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW146P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (epoetin) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching epoetin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising epoetin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and epoetin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Epoetin preferably is covalently attached to a side chain, the N-terminus or the C- terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide. CW146P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting epoetin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering epoetin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, epoetin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, epoetin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and epoetin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, epoetin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, epoetin is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW146P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching epoetin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, epoetin and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize epoetin and prevent its digestion in the stomach. In addition, CW146P

the pharmacologic effect can be prolonged by delayed release of epoetin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Epoetin is the subject of EP 148605 B ( 1990), priority US application 561024

(1983), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises epoetin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW146P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be CW146P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, l.ow molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier poljφeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW146P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW146P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

11 CW146P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, epoetin is covalently attached to the polypeptide via a peptide linkage.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW146P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the ttansported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzj ie for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-epoetin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW146P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW146P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW146P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and epoetin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said poljφeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said poljφeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein epoetin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW146P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein epoetin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing epoetin from said composition in a pH-dependent manner.

19. A method for protecting epoetin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for confrolling release of epoetin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching epoetin to said polypeptide.

21. A method for delivering epoetin to a patient comprising administering to said patient a composition comprising: a polypeptide; and epoetin covalently attached to said polypeptide.

22. The method of claim 21 wherein epoetin is released from said composition by an enzyme-catalyzed release.

17 CW146P

23. The method of claim 21 wherein epoetin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW146P

Abstract

A composition comprising a polypeptide and epoetin covalently attached to the polypeptide. Also provided is a method for delivery of epoetin to a patient comprising administering to the patient a composition comprising a polypeptide and epoetin covalently attached to the polypeptide. Also provided is a method for protecting epoetin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of epoetin from a composition comprising covalently attaching it to the polypeptide.

19 CW147P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING EPTIFIBATIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to eptifibatide, as well as methods for protecting and administering eptifibatide. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Eptifibatide is a known phaimaceutical agent that is used in the treatment of thrombosis, angina, myocardial infarction and restenosis. Its chemical name is N6- (aminoiminomethyl)-N2-(3-mercapto- 1 -oxopropyl)-L-lysylglycyl-L-alph a-aspartyl-L- tryptophyl-L-prolyl-L-cysteinamide cyclic (l-6)-disulfide. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an¹ oral dosage form when none exists. The novel pharmaceutical CW147P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW147P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzjmies residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW147P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzjmies. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (eptifibatide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching eptifibatide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising eptifibatide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and eptifibatide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW147P

Eptifibatide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the poljφeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting eptifibatide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering eptifibatide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, eptifibatide is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, eptifibatide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and eptifibatide is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, eptifibatide is CW147P

released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, eptifibatide is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching eptifibatide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, eptifibatide and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW147P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize eptifibatide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of eptifibatide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Eptifibatide is the subject of U.S. Patent Numbers 5,686,570, 5,756,451 and 5,807,825, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises eptifibatide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture. CW147P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Tjφically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW147P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW147P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW147P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW147P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, eptifibatide is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW147P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-eptifibatide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW147P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-ter minus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW147P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW147P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW147P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and eptifibatide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said poljφeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein eptifibatide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW147P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein eptifibatide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing eptifibatide from said composition in a pH-dependent manner.

19. A method for protecting eptifibatide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of eptifibatide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching eptifibatide to said polypeptide.

21. A method for delivering eptifibatide to a patient comprising administering to said patient a composition comprising: a polypeptide; and eptifibatide covalently attached to said polypeptide.

22. The method of claim 21 wherein eptifibatide is released from said composition by an enzyme-catalyzed release.

18 CW147P

23. The method of claim 21 wherein eptifibatide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and eptifibatide covalently attached to the polypeptide. Also provided is a method for delivery of eptifibatide to a patient comprising administering to the patient a composition comprising a polypeptide and eptifibatide covalently attached to the polypeptide. Also provided is a method for protecting eptifibatide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of eptifibatide from a composition comprising covalently attaching it to the polypeptide.

19 CW148P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ERGOT AMINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to ergotamine, as well as methods for protecting and administering ergotamine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ergotamine is a known pharmaceutical agent that is used in the treatment of migraines. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW148P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating.matrix, which may not be quantitative and may complicate dosage CW148P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW148P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ergotamine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ergotamine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechamsm.

Altematively, the present invention provides a pharmaceutical composition comprising ergotamine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ergotamine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ergotamine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW148P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ergotamine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ergotamine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, ergotamine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, ergotamine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ergotamine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, ergotamine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ergotamine is released from the composition in a CW148P

sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a earner peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ergotamine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ergotamine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is CW148P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ergotamine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ergotamine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ergotamine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW148P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW148P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW148P

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW148P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW148P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ergotamine is covalently attached to the polypeptide via the alcohol or amine groups.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW148P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ergotamine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW148P

Acid/N-ter minus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopjτidine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW148P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW148P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ergotamine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said poljφeptide is a heteropoljmier of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein ergotamine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

1 1. The composition of claim 1 further comprising an adjuvant.

16 CW148P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ergotamine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ergotamine from said composition in a pH-dependent manner.

19. A method for protecting ergotamine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ergotamine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ergotamine to said polypeptide.

21. A method for delivering ergotamine to a patient comprising administering to said patient a composition comprising: a polypeptide; and ergotamine covalently attached to said polypeptide.

22. The method of claim 21 wherein ergotamine is released from said composition by an enzyme-catalyzed release.

17 CW148P

23. The method of claim 21 wherein ergotamine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW148P

Abstract

A composition comprising a polypeptide and ergotamine covalently attached to the polypeptide. Also provided is a method for delivery of ergotamine to a patient comprising administering to the patient a composition comprising a polypeptide and ergotamine covalently attached to the poljφeptide. Also provided is a method for protecting ergotamine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ergotamine from a composition comprising covalently attaching it to the polypeptide.

19 CW149P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ERYTHROMYCIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to eiythromycin, as well as methods for protecting and administering eiythromycin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Eiythromycin is a known pharmaceutical agent that is used in the treatment of bacterial infections. Its chemical name is (3R *, 4S*, 5S*, 6R *, 7R *, 9R * 1 IR *, 12R *, 13S* 14R*)-4-((2,6-Dideoxy-3-C-methyl-3-O-methyl-a-L- ribo- hexopyranosyl) -oxy) - 14- ethyl-7,12,13- trihydroxy - 3,5,7,9,11,13-hexa methyl-6- ((3,4,6-trideoxy-3- (dimethylamino)-b-D- y/o- hexopyran osyl)oxy)oxacyclotetradecane-2, 10-dione. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW149P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW149P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexfran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW149P

Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(eiythromycin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching eiythromycin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or poljφeptide, also refeoed to herein as a carrier peptide. In certain applications, the poljφeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising eiythromycin microencapsulated by a poljφeptide.

The invention provides a composition comprising a polypeptide and eiythromycin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Eiythromycin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW149P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting eiythromycin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering eiythromycin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, eiythromycin is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, erythromycin is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and erythromycin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, erythromycin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, erythromycin is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and CW149P

release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching erythromycin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, erythromycin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW149P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize erythromycin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of erythromycin. Furthermore^ active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises erythromycin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW149P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be CW149P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier poljφeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW149P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoπnational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW149P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

11 CW149P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-dmg dimer. This carrier peptide-dmg conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, erythromycin is covalently attached to the polypeptide via a hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW149P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asjmunetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-erythromycin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW149P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C- terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW149P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW149P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and erythromycin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein erythromycin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW149P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein erythromycin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing erythromycin from said composition in a pH-dependent manner.

19. A method for protecting erythromycin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of erythromycin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching erythromycin to said polypeptide.

21. A method for delivering erythromycin to a patient comprising administering to said patient a composition comprising: a polypeptide; and erythromycin covalently attached to said polypeptide.

22. The method of claim 21 wherein erythromycin is released from said composition by an enzyme-catalyzed release.

17 CW149P

23. The method of claim 21 wherein erythromycin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW149P

Abstract

A composition comprising a polypeptide and erythromycin covalently attached to the polypeptide. Also provided is a method for delivery of erythromycin to a patient comprising administering to the patient a composition comprising a polypeptide and erythromycin covalently attached to the polypeptide. Also provided is a method for protecting erythromycin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of erythromycin from a composition comprising covalently attaching it to the polypeptide.

19 CW150P

A NOVEL PHARMACEUTICAL COMPOUND AND METHODS OF MAKING

AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to advanced glycosylation endproduct (AGE) crosslink breaker, as well as methods for protecting and administering AGE crosslink breaker. This novel compound, refened to as a CARRIER WAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

The AGE crosslink breaker of the present invention is a known pharmaceutical agent that is used in the treatment of diabetes and cardiovascular disease. Its chemical name is 4,5-dimethyl-3-(2-oxo-2-phenylethyl)thiazolium. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW150P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW150P

reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW150P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (AGE crosslink breaker) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching AGE crosslink breaker to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising AGE crosslink breaker microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and AGE crosslink breaker covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

AGE crosslink breaker preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active CW150P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting AGE crosslink breaker from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering AGE crosslink breaker to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, AGE crosslink breaker is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, AGE crosslink breaker is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and AGE crosslink breaker is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, AGE crosslink breaker is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, AGE CW150P

crosslink breaker is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching AGE crosslink breaker to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, AGE crosslink breaker and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW150P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize AGE crosslink breaker and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of AGE crosslink breaker. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

AGE crosslink breaker is the subject of WO 96/22095 (1996), priority US application 375155 (1995), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises AGE crosslink breaker covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding CW150P

are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW150P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW150P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW150P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-dmg dimer can be converted into the gamma ester of glutamic acid N-

11 CW150P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW150P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asjm metrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide- AGE crosslink breaker conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW150P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-ter minus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-ter minus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW150P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW150P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW150P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and AGE crosslink breaker covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein AGE crosslink breaker is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW150P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein AGE crosslink breaker is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing AGE crosslink breaker from said composition in a pH-dependent manner.

19. A method for protecting AGE crosslink breaker from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of AGE crosslink breaker from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching AGE crosslink breaker to said polypeptide.

21. A method for delivering AGE crosslink breaker to a patient comprising administering to said patient a composition comprising: a polypeptide; and AGE crosslink breaker covalently attached to said polypeptide.

22. The method of claim 21 wherein AGE crosslink breaker is released from said composition by an enzyme-catalyzed release.

18 CW150P

23. The method of claim 21 wherein AGE crosslink breaker is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and AGE crosslink breaker covalently attached to the polypeptide. Also provided is a method for delivery of AGE crosslink breaker to a patient comprising administering to the patient a composition comprising a polypeptide and AGE crosslink breaker covalently attached to the polypeptide. Also provided is a method for protecting AGE crosslink breaker from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of AGE crosslink breaker from a composition comprising covalently attaching it to the polypeptide.

19 CW151P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ESATENOLOL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to esatenolol, as well as methods for protecting and administering esatenolol. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Esatenolol is a known pharmaceutical agent that is used in the tteatment of hypertension. Its chemical name is (S)-4-[2-hydroxy-3-[(l-methylethyl)amino] propoxy]benzeneacetamide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken CW151P

under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzjmie degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent CW151P

in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR CW151P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (esatenolol) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching esatenolol to the N- teoninus, the C-teoninus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the poljφeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising esatenolol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and esatenolol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Esatenolol preferably is covalently attached to a side chain, the N-teoninus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is CW151P

an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting esatenolol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering esatenolol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, esatenolol is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, esatenolol is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and esatenolol is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, esatenolol is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, esatenolol is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW151P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching esatenolol to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, esatenolol and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW151P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize esatenolol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of esatenolol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Esatenolol is the subject of GB 1285035 (1972), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises esatenolol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are pol miers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW151P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW151P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW151P

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 - Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW151P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- teπninus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples,^" the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW151P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechamsm can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, esatenolol is covalently attached to the polypeptide via the amido group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW151P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-esatenolol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW151P

Acid N- terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW151P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW151P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and esatenolol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein esatenolol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW151P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein esatenolol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing esatenolol from said composition in a pH-dependent manner.

19. A method for protecting esatenolol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for confrolling release of esatenolol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching esatenolol to said polypeptide.

21. A method for delivering esatenolol to a patient comprising administering to said patient a composition comprising: a polypeptide; and esatenolol covalently attached to said polypeptide.

22. The method of claim 21 wherein esatenolol is released from said composition by an enzyme-catalyzed release.

17 CW151P

23. The method of claim 21 wherein esatenolol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW151P

Abstract

A composition comprising a polypeptide and esatenolol covalently attached to the polypeptide. Also provided is a method for delivery of esatenolol to a patient comprising administering to the patient a composition comprising a polypeptide and esatenolol covalently attached to the polypeptide. Also provided is a method for protecting esatenolol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for conttolling release of esatenolol from a composition comprising covalently attaching it to the polypeptide.

19 CW152P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ESTERIFIED ESTROGENS AND METHYLTESTOSTERONE AND METHODS

OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to esterified esfrogens and methyltestosterone, as well as methods for protecting and administering esterified esfrogens and methyltestosterone. This novel compound, refened to as a C ARRIERW AVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Esterified estrogens and methyltestosterone are known pharmaceutical agents that are used together in hormone replacement therapy. Esterified estrogens is a mixture of the sodium salts of the sulfate esters of the esfrogenic substances, principally estrone, that are of the type excreted in the urine of pregnant mares. Estrone sodium sulfate is the principal active ingredient in esterified esfrogens. Esterified esfrogens may be derived from natural sources and/or prepared synthetically. The stracture of estrone is:

CW152P

The structure of methyltestosterone is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the CW152P

active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the CW152P

gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (xxxxx) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching esterified esfrogens and methyltestosterone to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active CW152P

agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising esterified estrogens and methyltestosterone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and esterified esfrogens and methyltestosterone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Xxxxx preferably is covalently attached to a side chain, the N-terminus or the C- terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a - microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter. CW152P

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting esterified estrogens and methyltestosterone from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering esterified estrogens and methyltestosterone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, esterified estrogens and methyltestosterone is released from the composition by an enzyme- catalyzed release. In another prefened embodiment, esterified estrogens and methyltestosterone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and esterified esfrogens and methyltestosterone is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, esterified estrogens and methyl t&tosterone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, esterified estrogens and methyltestosterone is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: CW152P

(a) attaching esterified estrogens and methyltestosterone to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, esterified estrogens and methyltestosterone and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize esterified estrogens and methyltestosterone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of esterified estrogens and methyltestosterone. Furthermore, active CW152P

agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises esterified esfrogens and methyltestosterone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the poljφeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW152P

and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will CW152P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of die peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the

10 CW152P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g.„ aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order

11 CW152P

to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig.4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

12 CW152P

preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the poljφeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, esterified estrogens and methyltestosterone are covalently attached to the polypeptide via their hydroxy groups.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Altematively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

13 CW152P

intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enz mie for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-xxxxx conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

14 CW152P

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be freated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or ttibutylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl Glutamate/C-Terminus Conjugation

15 CW152P

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly.γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW152P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and xxxxx covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein esterified estrogens and methyltestosterone is covalently attached to a side chain, the N-terminus or the C- terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW152P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein esterified estrogens and methyltestosterone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing esterified esfrogens and methyltestosterone from said composition in a pH-dependent manner.

19. A method for protecting esterified estrogens and methyltestosterone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of esterified estrogens and methyltestosterone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching esterified esfrogens and methyltestosterone to said polypeptide.

21. A method for delivering esterified esfrogens and methyltestosterone to a patient comprising administering to said patient a composition comprising: a polypeptide; and xxxxx covalently attached to said polypeptide.

18 CW152P

22. The method of claim 21 wherein esterified estrogens and methyltestosterone s released from said composition by an enzyme-catalyzed release.

23. The method of claim 21 wherein esterified esfrogens and methyltestosterone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and esterified estrogens and methyltestosterone covalently attached to the polypeptide. Also provided is a method for delivery of esterified estrogens and methyltestosterone to a patient comprising administering to the patient a composition comprising a polypeptide and esterified estrogens and methyltestosterone covalently attached to the polypeptide. Also provided is a method for protecting esterified estrogens and methyltestosterone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of esterified esfrogens and methyltestosterone from a composition comprising covalently attaching it to the polypeptide.

19 CW153P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING

CONJUGATED ESTROGENS AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to conjugated estrogens, as well as methods for protecting and administering conjugated esfrogens. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining t with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

In women, oral conjugated estrogens USP and synthetic conjugated esfrogens A are used for the management of moderate to severe vasomotor symptoms associated with menopause. Conjugated estrogens USP is a mixture containing the sodium salts of the water-soluble sulfate esters of estrone and equilin derived wholly or in part from equine urine or may be prepared synthetically from estrone and equilin. Conjugated esfrogens USP also contains conjugated esfrogenic substances of the type excreted by pregnant mares including 17alpha-dihydroequilin, 17alpha-esfradiol, 17beta-dihydroequilin, equilenin, 17alpha-dihydroequilenin, 17beta-dihydroequilenin, Delta^8,9-dehydroestrone, and 17beta-esfradiol. Conjugated estrogens USP contains 52.5 — 61.5% sodium estrone sulfate and 22.5 — 30.5% sodium equilin sulfate. Conjugated esfrogens contains, as sodium sulfate conjugates, 13.5 — 19.5% 17alpha-dihydroequilin, 2.5 — 9.5% 17alpha- esfradiol, and 0.5 — 4% 17beta-dihydroequilin. They are available from natural sources. The stracture of estrone is: CW153P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and fransport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the CW153P

active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the CW153P

gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in tum, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent ( conjugated estrogens) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching conjugated estrogens to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a- carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active CW153P

agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising conjugated estrogens microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and conjugated estrogens covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring ammo acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

conjugated estrogens preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the poljφeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be CW153P

conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting conjugated estrogens from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering conjugated esfrogens to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, conjugated estrogens are released from the composition by an enzyme-catalyzed release. In another prefened embodiment, conjugated esfrogens are released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and conjugated estrogens are released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, conjugated estrogens are released from the composition by a pH- dependent unfolding of the polypeptide. In another preferred embodiment, conjugated estrogens are released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching conjugated estrogens to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and CW153P

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, conjugated estrogens and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize conjugated esfrogens and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of conjugated estrogens. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises conjugated estrogens covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a CW153P

homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal CW153P

packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of CW153P

the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzjmiatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

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TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or poljφeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

u CW153P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW153P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, conjugated esfrogens are covalently attached to the polypeptide via the hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW153P

mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide- conjugated estrogens conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AcidTN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Anune/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

14 CW153P

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hyciroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylammopyridine, triethylamine or tributylamine.

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW153P

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW153P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and conjugated esfrogens covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein conjugated estrogens are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW153P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein conjugated estrogens are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing conjugated estrogens from said composition in a pH-dependent manner.

19. A method for protecting conjugated estrogens from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of conjugated estrogens from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching conjugated estrogens to said polypeptide.

21. A method for delivering conjugated estrogens to a patient comprising administering to said patient a composition comprising: a polypeptide; and conjugated estrogens covalently attached to said polypeptide.

22. The method of claim 21 wherein conjugated esfrogens are released from said composition by an enzyme-catalyzed release.

18 CW153P

23. The method of claim 21 wherein conjugated esfrogens are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and conjugated estrogens covalently attached to the polypeptide. Also provided is a method for delivery of conjugated estrogens to a patient comprising administering to the patient a composition comprising a poljφeptide and conjugated estrogens covalently attached to the polypeptide. Also provided is a method for protecting conjugated esfrogens from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of conjugated estrogens from a composition comprising covalently attaching it to the polypeptide.

19 CW154P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING

CONJUGATED ESTROGENS AND MEDROXYPROGESTERONE ACETATE

AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to conjugated esfrogens and medroxyprogesterone acetate, as well as methods for protecting and administering conjugated estrogens and medroxyprogesterone acetate. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combimng it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Conjugated estrogens and medroxjφrogesterone acetate are known pharmaceutical agents used in hormone replacement therapy.

In women, oral conjugated estrogens USP and synthetic conjugated estrogens A are used for the management of moderate to severe vasomotor symptoms associated with menopause. Conjugated estrogens USP is a mixture containing the sodium salts of the water-soluble sulfate esters of estrone and equilin derived wholly or in part from equine urine or may be prepared synthetically from estrone and equilin. Conjugated esfrogens USP also contains conjugated esfrogenic substances of the type excreted by pregnant mares including 17alpha-dihydroequilin, 17alpha-esfradiol, 17beta-dihydroequilin, ft 0 equilenin, 17alpha-dihydroequilenin, 17beta-dihydroequilenin, Delta ' -dehydroesfrone, and 17beta-estradiol. Conjugated estrogens USP contains 52.5 — 61.5% sodium estrone sulfate and 22.5 — 30.5% sodium equilin sulfate. Conjugated esfrogens contains, as sodium sulfate conjugates, 13.5 — 19.5% 17alpha-dihydroequilin, 2.5 — 9.5% 17alpha- esttadiol, and 0.5 — 4% 17beta-dihydroequilin. They are available from natural sources. The structure of estrone is: CW154P

Medroxyprogesterone acetate is a synthetic progestin. Medroxyprogesterone acetate is a derivative of 17 alpha-hydroxyprogesterone that differs structurally by the addition of a 6 alpha-methyl group and a 17 alpha-acetate group. Its structure is:

The novel pha naceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase CW154P

markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzjmies in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzjmie degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzjmie degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large a ay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some CW154P

technologies, control of the degradation process required for active agent release is unreliable: For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR CW154P

application, but absoφtion through the bmsh-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (esfrogens and medroxyprogesterone acetate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching conjugated estrogens and medroxyprogesterone acetate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising conjugated estrogens and medroxyprogesterone acetate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and conjugated estrogens and medroxyprogesterone acetate covalently attached to the polypeptide.

Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Estrogens and medroxyprogesterone acetate preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N- CW154P

terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C- terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting conjugated estrogens and medroxyprogesterone acetate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering conjugated estrogens and medroxyprogesterone acetate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, conjugated estrogens and medrox φrogesterone acetate are released from the composition by an enzyme-catalyzed release. In another prefened embodiment, conjugated estrogens and medroxyprogesterone acetate are released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and conjugated estrogens and medroxyprogesterone acetate are released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, conjugated estrogens and CW154P

medroxyprogesterone acetate are released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, conjugated estrogens and medroxyprogesterone acetate are released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching conjugated estrogens and medroxyprogesterone acetate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, conjugated estrogens and medroxyprogesterone acetate and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW154P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize conjugated esfrogens and medroxyprogesterone acetate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of conjugated estrogens and medroxyprogesterone acetate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises conjugated estrogens and medroxyprogesterone acetate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the djmamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding CW154P

are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW154P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophiUcity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the

10 CW154P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

11 CW154P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N-

12 CW154P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, conjugated esfrogens and medroxyprogesterone acetate are covalently attached to the polypeptide via the hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

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The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system, will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-esttogens and medroxyprogesterone acetate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

14 CW154P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Aπύne/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

15 CW154P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylajninopyridine, triethylamine or ttibutylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

16 ²⁹⁵⁸ CW154P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and estrogens and medroxyprogesterone acetate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said poljφeptide is a heteropoljmier of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein conjugated estrogens and medroxyprogesterone acetate are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

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10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein conjugated estrogens and medroxyprogesterone acetate are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing conjugated estrogens and medroxyprogesterone acetate from said composition in a pH- dependent manner.

19. A method for protecting conjugated estrogens and medroxyprogesterone acetate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of conjugated estrogens and medroxyprogesterone acetate from a composition wherein said composition comprises a

18 CW154P

polypeptide, said method comprising covalently attaching conjugated estrogens and medroxyprogesterone acetate to said polypeptide.

21. A method for delivering conjugated estrogens and medroxyprogesterone acetate to a patient comprising administering to said patient a composition comprising: a polypeptide; and esfrogens and medroxyprogesterone acetate covalently attached to said polypeptide.

22. The method of claim 21 wherein conjugated estrogens and medroxyprogesterone acetate are released from said composition by an enzjmie-catalyzed release.

23. The method of claim 21 wherein conjugated estrogens and medroxyprogesterone acetate are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and conjugated estrogens and medroxyprogesterone acetate covalently attached to the polypeptide. Also provided is a method for delivery of conjugated estrogens and medroxyprogesterone acetate to a patient comprising administering to the patient a composition comprising a polypeptide and conjugated esfrogens and medroxyprogesterone acetate covalently attached to the polypeptide. Also provided is a method for protecting conjugated estrogens and medroxyprogesterone acetate from degradation comprising covalently attaching it to a polypeptide.

19 CW155P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ESTROPIPATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to esfropipate, as well as methods for protecting and administering esfropipate. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Esfropipate is a known pharmaceutical agent that is used in hormone replacement therapy. Esfropipate is estrone solubilized as the sulfate and stabilized with piperazine. Conjugation of estrone with sulfate at the 3-hydroxy position on ring A of the steroid nucleus results in the formation of a water-soluble derivative; the pharmacologically inert piperazine moiety acts as a buffer to increase the stability and uniform potency of estrone sulfate. The stracture of estrone is:

CW155P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW155P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in tum, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW155P

linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested* almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (esfropipate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching esfropipate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is

^* controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising estropipate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and estropipate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, CW155P

(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Esfropipate preferably is covalently attached to a side chain, the N-terminus or die C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting esfropipate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering estropipate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the CW155P

polypeptide. In a prefened embodiment, esfropipate is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, esfropipate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and esfropipate is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, estropipate is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, esfropipate is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching estropipate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, estropipate and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the poljφeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side CW155P

chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize esfropipate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of estropipate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises esfropipate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture. CW155P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or confomiation processes. CW155P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

• Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW155P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW155P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW155P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Altematively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, estropipate is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW155P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-esttopipate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW155P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be freated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW155P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW155P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW155P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and estropipate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein esfropipate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW155P

12. The composition of claim 1 1 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein esfropipate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing esfropipate from said composition in a pH-dependent manner.

19. A method for protecting estropipate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of estropipate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching esfropipate to said polypeptide.

21. A method for delivering estropipate to a patient comprising administering to said patient a composition comprising: a polypeptide; and esfropipate covalently attached to said polypeptide.

22. The method of claim 21 wherein estropipate is released from said composition by an enzyme-catalyzed release.

18 CW155P

23. The method of claim 21 wherein estropipate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and estropipate covalently attached to the poljφeptide. Also provided is a method for delivery of esfropipate to a patient comprising administering to the patient a composition comprising a polypeptide and estropipate covalently attached to the polypeptide. Also provided is a method for protecting esfropipate from degradation comprising covalently attaching it to a poljφeptide. Also provided is a method for confrolling release of estropipate from a composition comprising covalently attaching it to the polypeptide.

19 CW156P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ETANERCEPT AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to etanercept, as well as methods for protecting and administering etanercept. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Etanercept is a known pharmaceutical agent that is used in the treatment of arthritis. Its chemical name is 1 -235-tumor necrosis factor receptor (human) fusion protein with 236-467-immunoglobulin GI (human gamma 1 -chain Fc fragment)

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW156P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and fransport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW156P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexfran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW156P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (etanercept) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching etanercept to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Altematively, the present invention provides a pharmaceutical composition comprising etanercept microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and etanercept covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Etanercept preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide. CW156P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting etanercept from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering etanercept to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefened embodiment, etanercept is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, etanercept is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and etanercept is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, etanercept is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, etanercept is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. . CW156P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching etanercept to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, etanercept and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a sjmthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize etanercept and prevent its digestion in the stomach. In CW156P

addition, the pharmacologic effect can be prolonged by delayed release of etanercept. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Etanercept is the subject of EP 418014 B (1995), priority based on US application

403241 (1989), herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises etanercept covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occvnring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary structure and the spatial arrangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW156P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be CW156P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same canier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level ofconformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate CW156P

weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several arninopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW156P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terrninus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to riOlyfhydroxypropylgluttunine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

11 CW156P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, etanercept is covalently attached to the polypeptide via a peptide bond.

The polypeptide canier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW156P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-etanercept conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product

13 CW156P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be freated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW156P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl GIutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW156P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and etanercept covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein etanercept is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW156P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein etanercept is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing etanercept from said composition in a pH-dependent manner.

19. A method for protecting etanercept from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of etanercept from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching etanercept to said polypeptide.

21. A method for delivering etanercept to a patient comprising administering to said patient a composition comprising: a polypeptide; and etanercept covalently attached to said polypeptide.

22. The method of claim 21 wherein etanercept is released from said composition by an enzyme-catalyzed release.

17 CW156P

⁴ 23. The method of claim 21 wherein etanercept is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW156P

Abstract

A composition comprising a polypeptide and etanercept covalently attached to the poljφeptide. Also provided is a method for delivery of etanercept to a patient comprising administering to the patient a composition comprising a polypeptide and etanercept covalently attached to the polypeptide. Also provided is a method for protecting etanercept from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of etanercept from a composition comprising covalently attaching it to the polypeptide.

19 CW157P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ETHINYL ESTRADIOL AND NORETHINDRONE AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ethinyl estradiol and norethindrone, as well as methods for protecting and administering ethinyl estradiol and norethindrone. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ethinyl estradiol and norethindrone are known pharmaceutical agents that are used together as a contraceptive. The stracture of ethinylestradiol is:

The stracture of norethindrone is:

CW157P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzjmies in the GI fract, permeability of cellular membranes and fransport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW157P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from, several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW157P

linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a poljφeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexfran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ethinyl estradiol and norethindrone) to a polymer ofpeptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ethinyl estradiol and norethindrone to the N-terminus, the C-terrninus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ethinyl estradiol and norethindrone microencapsulated by a polypeptide. CW157P

The invention provides a composition comprising a polypeptide and ethinyl estradiol and norethindrone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ethinyl estradiol and norethindrone preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ethinyl estradiol and norethindrone from degradation comprising covalently attaching it to a polypeptide. CW157P

The invention also provides a method for delivering ethinyl estradiol and norethindrone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, ethinyl estradiol and norethindrone are released from the composition by an enzyme-catalyzed release. In another prefened embodiment, ethinyl estradiol and norethindrone are released in a time- dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and ethinyl estradiol and norethindrone are released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, ethinyl estradiol and norethindrone are released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, ethinyl estradiol and norethindrone are released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ethinyl estradiol and norethindrone to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ethinyl estradiol and norethindrone and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis CW157P

of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ethinyl estradiol and norethindrone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ethinyl estradiol and norethindrone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ethinyl estradiol and norethindrone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetie amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW157P

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary stracture and the spatial arrangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent. CW157P

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide. CW157P

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights ofthe carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weigh, active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight ofthe polypeptide can be optimized depending on the level ofconformational protection desired. This property can be optimized in concert with the kinetics ofthe first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight ofthe carrier polypeptide. Another, significant advantage ofthe invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis ofthe key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 ofthe total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight ofthe carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

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TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% ofthe total weight ofthe active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain ofthe oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus ofthe oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

U CW157P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus ofthe peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus ofthe peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus ofthe peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide canier. The newly formed free amine ofthe glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading ofthe carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutaπuc acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other pol φeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue ofthe fact that the primary release ofthe drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

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The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ethinyl estradiol and norethindrone are covalently attached to the polypeptide via the hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment ofthe invention, a pre-first order release ofthe active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation ofthe membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties ofthe fransported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW157P

mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment ofthe invention, the composition includes one or more adjuvants to enhance the bioavailability ofthe active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion ofthe peptides.

Preferably, the resultant peptide-ethinyl estradiol and norethindrone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions ofthe invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

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Alcohol/N-Terminus Conjugation

In the following example the combination ofthe alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus ofthe peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be freated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The prodact is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW157P

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents ofthe claims and without departing from the spirit ofthe invention.

16 CW157P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ethinyl estradiol and norethindrone covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said poljφeptide is a heteropoljmier of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ethinyl estradiol and norethindrone are covalently attached to a side chain, the N-teiminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW157P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ethinyl estradiol and norethindrone are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ethinyl estradiol and norethindrone from said composition in a pH-dependent manner.

19. A method for protecting ethinyl estradiol and norethindrone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ethinyl estradiol and norethindrone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ethinyl esttadiol and norethindrone to said polypeptide.

21. A method for delivering ethinyl estradiol and norethindrone to a patient comprising administering to said patient a composition comprising: a polypeptide; and ethinyl estradiol and norethindrone covalently attached to said polypeptide.

22. The method of claim 21 wherein ethinyl estradiol and norethindrone are released from said composition by an enzyme-catalyzed release.

18 CW157P

23. The method of claim 21 wherein ethinyl esfradiol and norethindrone are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and ethinyl estradiol and norethindrone covalently attached to the polypeptide. Also provided is a method for delivery of ethinyl estradiol and norethindrone to a patient comprising administering to the patient a composition comprising a polypeptide and ethinyl esfradiol and norethindrone covalently attached to the polypeptide. Also provided is a method for protecting ethinyl estradiol and norethindrone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ethinyl estradiol and norethindrone from a composition comprising covalently attaching it to the poljφeptide.

19 CW157P

23. The method of claim 21 wherein ethinyl estradiol and norethindrone are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and ethinyl estradiol and norethindrone covalently attached to the polypeptide. Also provided is a method for delivery of ethinyl estradiol and norethindrone to a patient comprising administering to the patient a composition comprising a polypeptide and ethinyl estradiol and norethindrone covalently attached to the polypeptide. Also provided is a method for protecting ethinyl estradiol and norethindrone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ethinyl estradiol and norethindrone from a composition comprising covalently attaching it to the polypeptide.

19 CW158P

A NOVEL PHARMACEUTICAL COMPOUND AND METHODS OF MAKING

AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ACE /neutral endopeptidase inhibitor, as well as methods for protecting and administering ACE /neutral endopeptidase inhibitor. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment ofthe phaimaceutical market, and combining it with a carrier compound that enhances the usefulness ofthe pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

ACE /neutral endopeptidase inhibitor is a known pharmaceutical agent that is used in the treatment of hjφertension and heart failure. Its chemical name is [S-(R*,R*)]- hexahydro-6-[(2-mercapto-l-oxo-3-phenylpropyl)amino]-2,2-dimethyl-7-oxo-lH- azepine. Its stracture is:

The novel pharmaceutical compound ofthe present invention is useful in accomplishing one or more ofthe following goals: enhancement ofthe chemical stability ofthe original compound; alteration ofthe release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more ofthe following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW158P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability ofthe active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and fransport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzjmie degradation ofthe active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release ofthe active agent. For example, formulating diazepam. with a copolymer of glutamic acid and aspartic acid enables a sustained release ofthe active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation ofthe active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration ofthe active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW158P

reproducibility. In addition, encapsulated drags rely on diffusion out ofthe matrix, which is highly dependant on the water solubility ofthe active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control ofthe degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release ofthe drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size ofthe active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW158P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane ofthe intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment ofthe active agent (ACE /neutral endopeptidase inhibitor) to a polymer ofpeptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ACE /neutral endopeptidase inhibitor to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery ofthe active agent is controlled, in part, by the kinetics of unfolding ofthe carrier peptide. Upon entry into the upper intestinal fract, indigenous enzjmies release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds ofthe carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ACE /neutral endopeptidase inhibitor microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ACE /neutral endopeptidase inhibitor covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW158P

ACE /neutral endopeptidase inhibitor preferably is covalently attached to a side chain, the N-terminus or the C-terminus ofthe polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus ofthe polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus ofthe polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus ofthe polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus ofthe polypeptide.

The composition ofthe invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition ofthe invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding ofthe poljφeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ACE /neutral endopeptidase inhibitor from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ACE /neutral endopeptidase inhibitor to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, ACE /neutral endopeptidase inhibitor is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, ACE /neutral endopeptidase inhibitor is released in a time-dependent manner based on the pharmacokinetics ofthe enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and ACE /neutral endopeptidase inhibitor is released from the CW158P

composition by dissolution ofthe microencapsulating agent. In another prefened embodiment, ACE /neutral endopeptidase inhibitor is released from the composition by a pH-dependent unfolding ofthe polypeptide. In another prefened embodiment, ACE /neutral endopeptidase inhibitor is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release ofthe adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ACE /neutral endopeptidase inhibitor to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ACE /neutral endopeptidase inhibitor and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis ofthe polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain ofthe amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW158P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, ofthe invention. The general applications of this invention to other active phaimaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ACE /neutral endopeptidase inhibitor and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ACE /neutral endopeptidase inhibitor. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion ofthe active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

ACE /neutral endopeptidase inhibitor is the subject of EP 599444 B (1998), priority based on US application 884664 (1992), herein incorporated by reference, which describes how to make that drag.

The composition ofthe invention comprises ACE /neutral endopeptidase inhibitor covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture ofthe protein is the local conformation ofthe polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations ofthe chain determine the spatial anangement ofthe molecule. The CW158P

folding ofthe secondary stracture and the spatial aoangement ofthe side chains constitute the tertiary stracture.

Proteins fold because ofthe dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition ofthe protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" ofthe packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability ofthe folded protein where the degree to which ideal packing occurs determines the degree of relative stability ofthe protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature ofthe protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and CW158P

at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection ofthe amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity ofthe polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking ofthe peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures ofthe polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights ofthe carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain CW158P

length and molecular weight ofthe polypeptide can be optimized depending on the level ofconformational protection desired. This property can be optimized in concert with the kinetics ofthe first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight ofthe carrier polypeptide. Another, significant advantage ofthe invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis ofthe key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight ofthe carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW158P

TABLE

Amino acid MW Active agent MW

Glycine 57, Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% ofthe total weight ofthe active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain ofthe oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus ofthe oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

11 CW158P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus ofthe peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus ofthe peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus ofthe peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis ofthe key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine ofthe glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading ofthe carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue ofthe fact that the primary release ofthe drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW158P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain ofthe polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ACE /neutral endopeptidase inhibitor is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment ofthe invention, a pre- first order release ofthe active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation ofthe membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties ofthe transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW158P

mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment ofthe invention, the composition includes one or more adjuvants to enhance the bioavailability ofthe active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion ofthe peptides.

Preferably, the resultant peptide- ACE /neutral endopeptidase inhibitor conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions ofthe invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AcidVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

14 CW158P

Alcohol/N-TermJnus Conjugation

In the following example the combination ofthe alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus ofthe peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW158P

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents ofthe claims and without departing from the spirit ofthe invention.

16 CW158P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ACE /neutral endopeptidase inhibitor covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ACE /neutral endopeptidase inhibitor is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW158P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ACE /neutral endopeptidase inhibitor is conformationally protected by folding of said poljφeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ACE /neutral endopeptidase inhibitor from said composition in a pH-dependent manner.

19. A method for protecting ACE /neutral endopeptidase inhibitor from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ACE /neutral endopeptidase inhibitor from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ACE /neutral endopeptidase inhibitor to said polypeptide.

21. A method for delivering ACE /neutral endopeptidase inhibitor to a patient comprising administering to said patient a composition comprising: a polypeptide; and

ACE /neutral endopeptidase inhibitor covalently attached to said polypeptide.

18 CW158P

22. The method of claim 21 wherein ACE /neutral endopeptidase inhibitor is released from said composition by an enzyme-catalyzed release.

23. The method of claim 21 wherein ACE /neutral endopeptidase inhibitor is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and ACE /neutral endopeptidase inhibitor covalently attached to the polypeptide. Also provided is a method for delivery of ACE /neutral endopeptidase inhibitor to a patient comprising administering to the patient a composition comprising a polypeptide and ACE /neutral endopeptidase inhibitor covalently attached to the polypeptide. Also provided is a method for protecting ACE /neutral endopeptidase inhibitor from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ACE /neutral endopeptidase inhibitor from a composition comprising covalently attaching it to the polypeptide.

19 CW159P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ETHINYL ESTRADIOL AND ETHYNODIOL DIACETATE AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ethinyl estradiol and ethynodiol diacetate, as well as methods for protecting and administering ethinyl estradiol and ethynodiol diacetate. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment ofthe pharmaceutical market, and combining it with a carrier compound that enhances the usefulness ofthe pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ethinyl estradiol and ethynodiol diacetate are known pharmaceutical agents that are used together as a contraceptive. Each is isolatable from natural sources, or alternatively synthesized, by those of skill in the art.

The novel pharmaceutical compound ofthe present invention is useful in accomplishing one or more ofthe following goals: enhancement ofthe chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more ofthe following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW159P

invasive technique. Increasing the stability ofthe active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation ofthe active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release ofthe active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release ofthe active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation ofthe active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation ofthe active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out ofthe matrix, which is highly dependant on the water solubility ofthe active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control ofthe degradation process required for active agent release is CW159P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release ofthe drug and, as such, are not used for oral" administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size ofthe active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caniers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brash-border membrane ofthe intestines is limited to less than 5 microns. CW159P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment ofthe active agent (ethinyl estradiol and ethynodiol diacetate) to a polymer ofpeptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ethinyl estradiol and ethynodiol diacetate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery ofthe active agent is controlled, in part, by the kinetics of unfolding ofthe carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds ofthe carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ethinyl estradiol and ethynodiol diacetate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ethinyl estradiol and ethynodiol diacetate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ethinyl estradiol and ethynodiol diacetate preferably is covalently attached to a side chain, the N-terminus or the C-terminus ofthe polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N- terminus ofthe polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus ofthe polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C- CW159P

terminus ofthe polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus ofthe polypeptide.

The composition ofthe invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition ofthe invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ethinyl estradiol and ethynodiol diacetate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ethinyl estradiol and ethynodiol diacetate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, ethinyl estradiol and ethynodiol diacetate are released from the composition by an enzyme- catalyzed release. In another prefened embodiment, ethinyl estradiol and ethynodiol diacetate are released in a time-dependent manner based on the pharmacokinetics ofthe enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and ethinyl estradiol and ethynodiol diacetate are released from the composition by dissolution ofthe microencapsulating agent. In another prefened embodiment, ethinyl esfradiol and ethynodiol diacetate are released from the composition by a pH -dependent unfolding ofthe polypeptide. In another prefened embodiment, ethinyl estradiol and ethynodiol diacetate are released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further CW159P

comprises an adjuvant covalently attached to the polypeptide and release ofthe adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a canier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ethinyl estradiol and ethynodiol diacetate to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ethinyl estradiol and ethynodiol diacetate and a second active agent can be copolymerized in step (c). In another prefened embodiment the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis ofthe polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain ofthe amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, ofthe invention. The general applications of this invention to other active pharmaceutical agents is CW159P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, S the invention can stabilize ethinyl estradiol and ethynodiol diacetate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ethinyl estradiol and ethynodiol diacetate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion ofthe active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active 0 agents to specifics sites of action.

The composition ofthe invention comprises ethinyl estradiol and ethynodiol diacetate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an . oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer S of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure ofthe protein is the 0 local conformation ofthe polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations ofthe chain determine the spatial arrangement ofthe molecule. The folding ofthe secondary stracture and the spatial anangement ofthe side chains constitute the tertiary structure.

5 Proteins fold because ofthe dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition ofthe protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW159P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" ofthe packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability ofthe folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature ofthe protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW159P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection ofthe amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity ofthe polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking ofthe peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights ofthe carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight ofthe polypeptide can be optimized depending on the level ofconformational protection desired. This property can be optimized in concert with the kinetics ofthe first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight ofthe carrier polypeptide. Another, significant advantage ofthe invention is that the kinetics of CW159P

active agent release is primarily controlled by the enzymatic hydrolysis ofthe key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight ofthe carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% ofthe total weight ofthe active agent

10 CW159P

delivery composition or over two times the maximum drug loading for dextran. This is only for an.N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain ofthe oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus ofthe oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to polyfhydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus ofthe peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus ofthe peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus ofthe peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis ofthe key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine ofthe glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW159P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading ofthe carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue ofthe fact that the primary release ofthe drug moiety relies on peptidases and not on esterases. Altematively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain ofthe polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ethinyl estradiol and ethynodiol diacetate are covalently attached to the polypeptide via the hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW159P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts/have been shown to prevent protein unfolding. In another embodiment ofthe invention, a pre- first order release ofthe active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation ofthe membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties ofthe transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment ofthe invention, the composition includes one or more adjuvants to enhance the bioavailability ofthe active agent Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion ofthe peptides.

Preferably, the resultant peptide-ethinyl estradiol and ethynodiol diacetate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW159P

Compositions ofthe invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-ierminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination ofthe alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus ofthe peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be freated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW159P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alk l Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Polyjγ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be Hmited to the details shown. Rather,

15 CW159P

various modifications may be made in the details within the scope and range of equivalents ofthe claims and without departing from the spirit ofthe invention.

What is claimed is:

1. A pharmaceutical composition comprising: . a polypeptide; and ethinyl estradiol and ethynodiol diacetate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ethinyl estradiol and ethynodiol diacetate are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

16 CW159P

9. The composition of claim 1 further comprising a microencapsulating agent.

10/ The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ethinyl estradiol and ethynodiol diacetate are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ethinyl estradiol and ethynodiol diacetate from said composition in a pH-dependent manner.

19. A method for protecting ethinyl estradiol and ethynodiol diacetate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for confrolling release of ethinyl estradiol and ethynodiol diacetate from a composition wherein said composition comprises a polypeptide, said method

17 CW159P

comprising covalently attaching ethinyl estradiol and ethynodiol diacetate to said polypeptide.

21. A method for delivering ethinyl estradiol and ethynodiol diacetate to a patient comprising administering to said patient a composition comprising:

S a polypeptide; and ethinyl estradiol and ethynodiol diacetate covalently attached to said polypeptide.

22. The method of claim 21 wherein ethinyl estradiol and ethynodiol diacetate are released from said composition by an enzyme-catalyzed release.

23. The method of claim 21 wherein ethinyl estradiol and ethynodiol diacetate 0 are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant S from said composition is controlled by said polypeptide.

18 CW159P

Abstract

A composition comprising a polypeptide and ethinyl estradiol and ethynodiol diacetate covalently attached to the polypeptide. Also provided is a method for delivery of ethinyl estradiol and ethynodiol diacetate to a patient comprising administering to the patient a composition comprising a polypeptide and ethinyl estradiol and ethynodiol diacetate covalently attached to the polypeptide. Also provided is a method for protecting ethinyl estradiol and ethynodiol diacetate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ethinyl estradiol and ethynodiol diacetate from a composition comprising covalently attaching it to the polypeptide.

19 CW160P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ETHINYL ESTRADIOL AND LEVONORGESTREL AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ethinyl estradiol and levonorgestrel, as well as methods for protecting and admimstering ethinyl estradiol and . levonorgestrel. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment ofthe pharmaceutical market, and combining it with a carrier compound that enhances the usefulness ofthe pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ethinyl estradiol and levonorgestrel are are known pharmaceutical agents that are used together as a contraceptive. Each is isolatable from natural sources, or alternatively synthesized, by those of skill in the art.

The novel pharmaceutical compound ofthe present invention is useful in accomplishing one or more ofthe following goals: enhancement ofthe chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more ofthe following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW160P

invasive technique. Increasing the stability ofthe active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation ofthe active agent. Enzjmie inhibiting adjuvants have also been used to prevent enzjmie degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release ofthe active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation ofthe active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration ofthe active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out ofthe matrix, which is highly dependant on the water solubility ofthe active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control ofthe degradation process required for active agent release is CW160P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of poly aspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size ofthe active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane ofthe intestines is limited to less than 5 microns. CW160P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment ofthe active agent (ethinyl estradiol and levonorgestrel) to a polymer ofpeptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ethinyl estradiol and levonorgestrel to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery ofthe active agent is controlled, in part, by the kinetics of unfolding ofthe carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds ofthe carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ethinyl estradiol and levonorgestrel microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ethinyl estradiol and levonorgestrel covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ethinyl estradiol and levonorgestrel preferably is covalently attached to a side chain, the N-terminus or the C-terminus ofthe polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus ofthe polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus ofthe polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus CW160P

ofthe polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus ofthe polypeptide.

The composition ofthe invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition ofthe invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding ofthe polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ethinyl estradiol and levonorgesfrel from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ethinyl estradiol and levonorgesfrel to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment ethinyl estradiol and levonorgesfrel are released from the composition by an enzyme-catalyzed release. In another prefened embodiment, ethinyl estradiol and levonorgestrel are released in a time- dependent manner based on the pharmacokinetics ofthe enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and ethinyl estradiol and levonorgesfrel are released from the composition by dissolution ofthe microencapsulating agent. In another prefened embodiment, ethinyl estradiol and levonorgestrel are released from the composition by a pH-dependent unfolding ofthe polypeptide. In another prefened embodiment, ethinyl estradiol and levonorgesfrel are released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release ofthe adjuvant from the composition is controlled by the CW160P

polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ethinyl estradiol and levonorgestrel to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from die active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-caiboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ethinyl estradiol and levonorgestrel and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis ofthe polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular fransamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain ofthe amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, ofthe invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW160P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ethinyl estradiol and levonorgestrel and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release ofethinyl estradiol and levonorgestrel. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion ofthe active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition ofthe invention comprises ethinyl estradiol and levonorgestrel covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure ofthe protein is the local conformation ofthe polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations ofthe chain determine the spatial anangement ofthe molecule. The folding ofthe secondary stracture and the spatial arrangement ofthe side chains constitute the tertiary structure.

Proteins fold because ofthe dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW160P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense ofhydrogen bonds with water. Water molecules are "pushed out" ofthe packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability ofthe folded protein where the degree to which ideal packing occurs determines the degree of relative stability ofthe protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature ofthe protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW160P

Selection ofthe amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity ofthe polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking ofthe peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures ofthe polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights ofthe carrier compound can have profound effects on the active agent release kinetics. As a result low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight ofthe polypeptide can be optimized depending on the level ofconformational protection desired. This property can be optimized in concert with the kinetics ofthe first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight ofthe carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis ofthe key bond between the carrier peptide and the active agent. CW160P

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight ofthe carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% ofthe total weight ofthe active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW160P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain ofthe oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus ofthe oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to polyfhydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus ofthe peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus ofthe peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus ofthe peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis ofthe key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine ofthe glutamic acid residue will then undergo an inframolecular fransamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW160P

maximum drug loading ofthe carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue ofthe fact that the primary release ofthe drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ethinyl estradiol and levonorgestrel are covalently attached to the polypeptide via the hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Altematively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment ofthe

12 CW160P

invention, a pre-first order release ofthe active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation ofthe membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties ofthe transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment ofthe invention, the composition includes one or more adjuvants to enhance the bioavailability ofthe active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion ofthe peptides.

Preferably, the resultant peptide-ethinyl esfradiol and levonorgesfrel conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions ofthe invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW160P

AcidVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can.then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination ofthe alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus ofthe peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be freated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW160P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents ofthe claims and without departing from the spirit ofthe invention.

15 CW160P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ethinyl estradiol and levonorgesfrel covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ethinyl estradiol and levonorgestrel are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW160P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ethinyl estradiol and levonorgestrel are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ethinyl estradiol and levonorgesfrel from said composition in a pH-dependent manner.

19. A method for protecting ethinyl estradiol and levonorgestrel from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release ofethinyl estradiol and levonorgestrel from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ethinyl esfradiol and levonorgestrel to said polypeptide.

21. A method for delivering ethinyl esfradiol and levonorgesfrel to a patient comprising administering to said patient a composition comprising: a polypeptide; and ethinyl estradiol and levonorgestrel covalently attached to said polypeptide.

22. The method of claim 21 wherein ethinyl esfradiol and levonorgesfrel are released from said composition by an enzyme-catalyzed release.

17 CW160P

23. The method of claim 21 wherein ethinyl esfradiol and levonorgestrel are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW160P

Abstract

A composition comprising a polypeptide and ethinyl esfradiol and levonorgestrel covalently attached to the polypeptide. Also provided is a method for delivery ofethinyl estradiol and levonorgestrel to a patient comprising administering to the patient a composition comprising a polypeptide and ethinyl estradiol and levonorgestrel covalently attached to the polypeptide. Also provided is a method for protecting ethinyl estradiol and levonorgestrel from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release ofethinyl estradiol and levonorgestrel from a composition comprising covalently attaching it to the polypeptide.

19 CW161P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ETHINYL ESTRADIOL AND NORETHINDRONE 28 AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ethinyl estradiol and norethindrone 28, as well as methods for protecting and administering ethinyl estradiol and norethindrone 28. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment ofthe pharmaceutical market, and combining it with a carrier compound that enhances the usefulness ofthe pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ethinyl estradiol and norethindrone 28 are are known pharmaceutical agents that are used together as a contraceptive. Each is isolatable from natural sources, or alternatively synthesized, by those of skill in the art.

The novel pharmaceutical compound ofthe present invention is useful in accomplishing one or more ofthe following goals: enhancement ofthe chemical stability ofthe original compound; alteration ofthe release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more ofthe following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW161P

invasive technique. Increasing the stability ofthe active agent such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation ofthe active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release ofthe active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation ofthe active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration ofthe active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility ofthe active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control ofthe degradation process required for active agent release is CW161P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release ofthe drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight molecular size and particle size ofthe active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brash-border membrane ofthe intestines is limited to less than 5 microns. CW161P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment ofthe active agent (ethinyl estradiol and norethindrone 28) to a polymer ofpeptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ethinyl estradiol and norethindrone 28 to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery ofthe active agent is controlled, in part, by the kinetics of unfolding ofthe carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds ofthe carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ethinyl estradiol and norethindrone 28 microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ethinyl estradiol and norethindrone 28 covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ethinyl estradiol and norethindrone 28 preferably is covalently attached to a side chain, the N-terminus or the C-terminus ofthe polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus ofthe polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus ofthe polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus CW161P

ofthe polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus ofthe polypeptide.

The composition ofthe invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition ofthe invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding ofthe polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ethinyl estradiol and norethindrone 28 from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ethinyl estradiol and norethindrone 28 to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, ethinyl estradiol and norethindrone 28 are released from the composition by an enzyme- catalyzed release. In another prefened embodiment, ethinyl estradiol and norethindrone 28 are released in a time-dependent manner based on the pharmacokinetics ofthe enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and ethinyl esfradiol and norethindrone 28 are released from the composition by dissolution ofthe microencapsulating agent. In another prefened embodiment, ethinyl estradiol and norethindrone 28 are released from the composition by a pH-dependent unfolding ofthe polypeptide. In another prefened embodiment, ethinyl esfradiol and norethindrone 28 are released from the composition in a sustained release. In yet another prefened embodiment, the composition further'^' comprises an adjuvant covalently attached to the polypeptide and release ofthe adjuvant CW161P

from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ethinyl estradiol and norethindrone 28 to a side chain of an amino acid tb . form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent ethinyl estradiol and norethindrone 28 and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis ofthe polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain ofthe amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, ofthe invention.

The general applications of this invention to other active pharmaceutical agents is ^'

( CW161P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ethinyl estradiol and norethindrone 28 and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release ofethinyl estradiol and norethindrone 28. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion ofthe active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition ofthe invention comprises ethinyl estradiol and norethindrone 28 covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure ofthe protein is the local conformation ofthe polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations ofthe chain determine the spatial arrangement ofthe molecule. The folding ofthe secondary stracture and the spatial arrangement ofthe side chains constitute the tertiary structure.

Proteins fold because ofthe dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition ofthe protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW161P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense ofhydrogen bonds with water. Water molecules are "pushed out" ofthe packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability ofthe folded protein where the degree to which ideal packing occurs determines the degree of relative stability ofthe protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature ofthe protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's ' CW161P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection ofthe amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity ofthe polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking ofthe peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures ofthe polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects onihe active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight ofthe polypeptide can be optimized depending on the level ofconformational protection desired. This property can be optimized in concert with the kinetics ofthe first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight ofthe carrier polypeptide. Another, significant advantage ofthe invention is that the kinetics of CW161P

active agent release is primarily controlled by the enzymatic hydrolysis ofthe key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight ofthe carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% ofthe total weight ofthe active agent

10 CW161P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain ofthe oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus ofthe oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus ofthe peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus ofthe peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus ofthe peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig.4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine ofthe glutamic acid residue will then undergo an inframolecular fransamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

I carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW161P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading ofthe carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue ofthe fact that the primary release ofthe drug moiety relies on peptidases and not on esterases. Altematively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain ofthe polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ethinyl estradiol and norethindrone 28 are covalently attached to the polypeptide via the hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Altematively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW161P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment ofthe invention, a pre-first order release ofthe active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation ofthe membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties ofthe transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment ofthe invention, the composition includes one or more adjuvants to enhance the bioavailability ofthe active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion ofthe peptides.

Preferably, the resultant peptide-ethinyl estradiol and norethindrone 28 conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW161P

Compositions ofthe invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination ofthe alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus ofthe peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW161P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW161P

various modifications may be made in the details within the scope and range of equivalents ofthe claims and without departing from the spirit ofthe invention.

16 CW161P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ethinyl estradiol and norethindrone 28 covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ethinyl esfradiol and norethindrone 28 are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW161P

12. The composition of claim 1 1 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ethinyl estradiol and norethindrone 28 are conformationally protected by folding of said polypeptide about said active agent

18. The composition of claim 1 wherein said polypeptide is capable of releasing ethinyl estradiol and norethindrone 28 from said composition in a pH-dependent manner.

19. A method for protecting ethinyl estradiol and norethindrone 28 from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for confrolling release ofethinyl estradiol and norethindrone 28 from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ethinyl estradiol and norethindrone 28 to said polypeptide.

21. A method for delivering ethinyl estradiol and norethindrone 28 to a patient comprising administering to said patient a composition comprising: a polypeptide; and ethinyl estradiol and norethindrone 28 covalently attached to said polypeptide.

18 CW161P

22. The method of claim 21 wherein ethinyl esfradiol and norethindrone 28 are released from said composition by an enzyme-catalyzed release.

23. The method of claim 21 wherein ethinyl estradiol and norethindrone 28 are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and ethinyl estradiol and norethindrone 28 covalently attached to the polypeptide. Also provided is a method for delivery of ethinyl estradiol and norethindrone 28 to a patient comprising administering to the patient a composition comprising a polypeptide and ethinyl estradiol and norethindrone 28 covalently attached to the polypeptide. Also provided is a method for protecting ethinyl esfradiol and norethindrone 28 from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for confrolling release ofethinyl estradiol and norethindrone 28 from a composition comprising covalently attaching it to the polypeptide.

19 CW162P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ETHINYL ESTRADIOL AND NORETHINDRONE ACETATE AND METHODS

OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ethinyl estradiol and norethindrone acetate, as well as methods for protecting and administering ethinyl estradiol and norethindrone acetate. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment ofthe pharmaceutical market, and combining it with a carrier compound that enhances the usefulness ofthe pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ethinyl estradiol and norethindrone acetate are are known pharmaceutical agents that are used together as a contraceptive. Each is isolatable from natural sources, or alternatively synthesized, by those of skill in the art.

The novel pharmaceutical compound ofthe present invention is useful in accomplishing one or more ofthe following goals: enhancement ofthe chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more ofthe following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW162P

invasive technique. Increasing the stability ofthe active agent such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation ofthe active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release ofthe active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation ofthe active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large array of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation ofthe active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out ofthe matrix, which is highly dependant on the water solubility ofthe active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control ofthe degradation process required for active agent releasβls CW162P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of poly aspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight molecular size and particle size ofthe active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexfran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients.

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane ofthe intestines isTimited

( to less than 5 microns. CW162P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment ofthe active agent (ethinyl estradiol and norethindrone acetate) to a polymer ofpeptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ethinyl estradiol and norethindrone acetate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery ofthe active agent is controlled, in part, by the kinetics of unfolding ofthe carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds ofthe carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ethinyl estradiol and norethindrone acetate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ethinyl esfradiol and norethindrone acetate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ethinyl estradiol and norethindrone acetate preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N- terminus ofthe polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus ofthe polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C- CW162P

terminus ofthe polypeptide. In yet another prefened embodiment the active agent is an alcohol and is covalently attached to the N-terminus o the polypeptide.

The composition ofthe invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition ofthe invention is in the form of an ingestable tablet an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding ofthe polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ethinyl estradiol and norethindrone acetate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ethinyl estradiol and norethindrone acetate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, ethinyl estradiol and norethindrone acetate are released from the composition by an enzyme- catalyzed release. In another prefened embodiment, ethinyl estradiol and norethindrone acetate are released in a time-dependent manner based on the pharmacokinetics ofthe enzyme-catalyzed release. In another prefened embodiment the composition further comprises a microencapsulating agent and ethinyl estradiol and norethindrone acetate are released from the composition by dissolution ofthe microencapsulating agent. In another prefened embodiment, ethinyl esfradiol and norethindrone acetate are released from the composition by a pH-dependent unfolding ofthe polypeptide. In another prefened embodiment, ethinyl estradiol and norethindrone acetate are released from the , composition in a sustained release. In yet another prefened embodiment, the CW162P

composition further comprises an adjuvant covalently attached to the polypeptide and release ofthe adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ethinyl estradiol and norethindrone acetate to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ethinyl estradiol and norethindrone acetate and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis ofthe polypeptide and wherein the active agent is released from^* the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain ofthe amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, ofthe invention. The general applications of this invention to other active pharmaceutical agents is CW162P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First the invention can stabilize ethinyl estradiol and norethindrone acetate and prevent it« digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release ofethinyl estradiol and norethindrone acetate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion ofthe active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition ofthe invention comprises ethinyl estradiol and norethindrone acetate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure ofthe protein is the local conformation ofthe polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations ofthe chain determine the spatial anangement ofthe molecule. The folding ofthe secondary stracture and the spatial anangement ofthe side chains constitute the tertiary stracture.

Proteins fold because ofthe dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition ofthe protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW162P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

5 The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with

10 protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense ofhydrogen bonds with water. Water molecules are "pushed out" ofthe packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability ofthe folded protein where the degree to which ideal l s packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released.

20 The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature ofthe protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact partial unfolding of a protein is

25 often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's t CW162P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection ofthe amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity ofthe polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking ofthe peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight ofthe polypeptide can be optimized depending on the level ofconformational protection desired. This property can be optimized in concert with the kinetics ofthe first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight ofthe carrier polypeptide. Another, significant advantage ofthe invention is that the kinetics of CW162P

active agent release is primarily controlled by the enzymatic hydrolysis ofthe key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 ofthe total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight ofthe carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% ofthe total weight ofthe active agent

10 CW162P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain ofthe oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus ofthe oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchlorofoonate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus ofthe peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus ofthe peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus ofthe peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis ofthe key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine ofthe glutamic acid residue will then undergo an inframolecular fransamination reaction, thereby, releasing the active agent with coincident formation of pyrpglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW162P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading ofthe carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue ofthe fact that the primary release ofthe drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain ofthe polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ethinyl estradiol and norethindrone acetate are covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW162P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment ofthe invention, a pre-first order release ofthe active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex,

PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties ofthe transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment ofthe invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ethinyl esfradiol and norethindrone acetate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW162P

Compositions ofthe invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Aci /N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amme/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination ofthe alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus ofthe peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW162P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ- Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly.γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW162P

various modifications may be made in the details within the scope and range of equivalents ofthe claims and without departing from the spirit ofthe invention.

16 CW162P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and

S ethinyl estradiol and norethindrone acetate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

0 4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of S two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ethinyl estradiol and norethindrone acetate are covalently attached to a side chain, the N-terminus or the C-terminus of said 0 polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

17 CW162P

11. The composition of claim 1 further comprising an adjuvant.

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable S excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

0 16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ethinyl estradiol and norethindrone acetate are conformationally protected by folding of said polypeptide about said active agent.

5 18. The composition of claim 1 wherein said polypeptide is capable of releasing ethinyl estradiol and norethindrone acetate from said composition in a pH-dependent manner.

19. A method for protecting ethinyl estradiol and norethindrone acetate from degradation comprising covalently attaching said active agent to a polypeptide.

0 20. A method for controlling release of ethinyl estradiol and norethindrone acetate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ethinyl esfradiol and norethindrone acetate to said polypeptide.

21. A method for delivering ethinyl estradiol and norethindrone acetate to.a 5 patient comprising administering to said patient a composition comprising:

18 CW162P

a polypeptide; and ethinyl estradiol and norethindrone acetate covalently attached to said polypeptide.

22. The method of claim 21 wherein ethinyl estradiol and norethindrone acetate are released from said composition by an enzyme-catalyzed release.

23. The method of claim 21 wherein ethinyl estradiol and norethindrone acetate are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and ethinyl estradiol and norethindrone acetate covalently attached to the polypeptide. Also provided is a method for delivery of ethinyl estradiol and norethindrone acetate to a patient comprising administering to the patient a composition comprising a polypeptide and ethinyl estradiol and norethindrone acetate covalently attached to the polypeptide. Also provided is a method for protecting ethinyl estradiol and norethindrone acetate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for confrolling release of ethinyl estradiol and norethindrone acetate from a composition comprising covalently attaching it to the polypeptide.

19 CW163P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ETHINYL ESTRADIOL AND NORGESTIMATE AND METHODS OF MAKING

AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ethinyl estradiol and norgestimate, as well as methods for protecting and administering ethinyl esfradiol and norgestimate. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ethinyl estradiol and norgestimate are are known pharmaceutical agents that are used together as a contraceptive. Each is isolatable from natural sources, or alternatively synthesized, by those of skill in the art.

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW163P

invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such a-» resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabUity of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW163P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW163P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ethinyl estradiol and norgestimate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ethinyl estradiol and norgestimate to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ethinyl estradiol and norgestimate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ethinyl esttadiol and norgestimate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropoljmier of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ethinyl estradiol and norgestimate preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus CW163P

of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ethinyl estradiol and norgestimate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ethinyl estradiol and norgestimate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, ethinyl estradiol and norgestimate are released from the composition by an enzyme-catalyzed release. In another prefened embodiment, ethinyl estradiol and norgestimate are released in a time- dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and ethinyl estradiol and norgestimate are released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, ethinyl estradiol and norgestimate are released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, ethinyl estradiol and norgestimate are released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the CW163P

polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ethinyl estradiol and norgestimate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ethinyl estradiol and norgestimate and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW163P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ethinyl estradiol and norgestimate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release ofethinyl estradiol and norgestimate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ethinyl estradiol and norgestimate covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW163P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW163P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition, of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the canier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW163P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention

₍ has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of. covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW163P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

u CW163P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ethinyl estradiol and norgestimate are covalentiy attached to the polypeptide via the hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW163P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ethinyl estradiol and norgestimate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW163P

AciάVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can-then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW163P

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl^'glutamates prepared any one of which may be suitable for the-drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW163P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and ethinyl estradiol and norgestimate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition pf claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ethinyl estradiol and norgestimate are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW163P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ethinyl esfradiol and norgestimate are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ethinyl estradiol and norgestimate from said composition in a pH-dependent manner.

19. A method for protecting ethinyl esttadiol and norgestimate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ethinyl estradiol and norgestimate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ethinyl estradiol and norgestimate to said polypeptide.

21. A method for delivering ethinyl estradiol and norgestimate to a patient comprising administering to said patient a composition comprising: a polypeptide; and ethinyl estradiol and norgestimate covalently attached to said polypeptide.

22. The method of claim 21 wherein ethinyl estradiol and norgestimate are released from said composition by an enzyme-catalyzed release.

17 CW163P

23. The method of claim 21 wherein ethinyl esfradiol and norgestimate are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

( 25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW163P

Abstract

A composition comprising a polypeptide and ethinyl estradiol and norgestimate covalently attached to the polypeptide. Also provided is a method for delivery ofethinyl estradiol and norgestimate to a patient comprising administering to the patient a composition comprising a polypeptide and ethinyl estradiol and norgestimate covalently attached to the polypeptide. Also provided is a method for protecting ethinyl estradiol and norgestimate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ethinyl esfradiol and norgestimate from a composition comprising covalently attaching it to the polypeptide.

19 CW164P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ETHINYL ESTRADIOL AND NORGESTREL AND METHODS OF MAKING

AND USING SAME

FTELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ethinyl estradiol and norgestrel, as well as methods for protecting and administering ethinyl estradiol and norgestrel. This . novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ethinyl estradiol and norgestrel are are known pharmaceutical agents that are used together as a contraceptive. Each is isolatable from natural sources, or alternatively synthesized, by those of skill in the art.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW164P

invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation ofthe active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW164P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzyπ_es in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW164P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ethinyl estradiol and norgestrel) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ethinyl esttadiol and norgestrel to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ethinyl esttadiol and norgestrel microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ethinyl estradiol and norgestrel covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ethinyl esttadiol and norgestrel preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus CW164P

of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ethinyl estradiol and norgestrel from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ethinyl estradiol and norgestrel to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, ethinyl estradiol and norgestrel are released from the composition by an enzyme-catalyzed release. In another prefened embodiment, ethinyl estradiol and norgestrel are released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ethinyl esttadiol and norgestrel are released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, ethinyl estradiol and norgestrel are released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, ethinyl estradiol and norgestrel are released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the CW164P

polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ethinyl estradiol and norgestrel to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ethinyl estradiol and norgestrel and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW164P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabihze ethinyl estradiol and norgesttel and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ethinyl estradiol and norgestrel. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ethinyl estradiol and norgestrel covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constimte the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW164P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW164P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophϋicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW164P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weighl of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW164P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampiciliin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutaπύc acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW164P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ethinyl estradiol and norgestrel are covalently attached to the polypeptide via the hydroxyl groups.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW164P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can exp ct that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ethinyl esttadiol and norgestrel conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW164P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW164P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the^* drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW164P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ethinyl estradiol and norgesttel covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ethinyl estradiol and norgestrel are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW164P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

(

5 14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an 0 oral suspension.

17. The composition of claim 1 wherein ethinyl esfradiol and norgestrel are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ethinyl esttadiol and norgestrel from said composition in a pH-dependent manner.

5 19. A method for protecting ethinyl estradiol and norgestrel from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of ethinyl estradiol and norgestrel from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ethinyl estradiol and norgesttel to said polypeptide.

0 21. A method for delivering ethinyl estradiol and norgestrel to a patient comprising administering to said patient a composition comprising: a polypeptide; and ethinyl estradiol and norgestrel covalently attached to said polypeptide.

22. The method of claim 21 wherein ethinyl estradiol and norgestrel are released 5 from said composition by an enzyme-catalyzed release.

17 CW164P

23. The method of claim 21 wherein ethinyl esttadiol and norgestrel are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW164P

Abstract

A composition comprising a polypeptide and ethinyl esttadiol and norgesttel covalently attached to the polypeptide. Also provided is a method for delivery ofethinyl estradiol and norgestrel to a patient comprising administering to the patient a composition comprising a polypeptide and ethinyl estradiol and norgestrel covalently attached to the polypeptide. Also provided is a method for protecting ethinyl esfradiol and norgestrel from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for conttolling release of ethinyl estradiol and norgestrel from a composition comprising covalently attaching it to the polypeptide.

19 CW169P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING EXENDIN-4 AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to exendin-4, as well as methods for protecting and administering exendin-4. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Exendin-4 is a known pharmaceutical agent that is used in the treatment of diabetes. It is a synthetic form of a peptide isolated from the salivary secretions of the Gila monster lizard.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified, when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW169P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW169P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW169P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (exendin- 4) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching exendin-4 to the N- terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising exendin-4 microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and exendin-4 covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Exendin-4 preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide. CW169P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationaUy protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting exendin-4 from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering exendin-4 to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, exendin-4 is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, exendin-4 is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and exendin-4 is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, exendin-4 is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, exendin-4 is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW169P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching eχendin-4 to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, exendin-4 and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize exendin-4 and prevent its digestion in the stomach. In CW169P

addition, the pharmacologic effect can be prolonged by delayed release of exendin-4. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises exendin-4 covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW169P

and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will CW169P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given appUcation. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the poljφeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage ofthe invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the CW169P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order

10 CW169P

to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Altematively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

n CW169P

preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, exendin-4 is covalently attached to the polypeptide via a peptide bond.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

12 CW169P

intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-exendin-4 conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

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The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodϋπύde or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

14 CW169P

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- lkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW169P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and exendin-4 covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein exendin-4 is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein exendin-4 is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing exendin-4 from said composition in a pH-dependent manner.

19. A method for protecting exendin-4 from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of exendin-4 from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching exendin-4 to said polypeptide.

21. A method for delivering exendin-4 to a patient comprising administering to said patient a composition comprising: a polypeptide; and exendin-4 covalently attached to said polypeptide.

22. The method of claim 21 wherein exendin-4 is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein exendin-4 is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW169P

Abstract

A composition comprising a polypeptide and exendin-4 covalently attached to the polypeptide. Also prαvided is a method for delivery of exendin-4 to a patient comprising administering to the patient a composition comprising a polypeptide and exendin-4 covalently attached to the polypeptide. Also provided is a method for protecting exendin- 4 from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for conttolling release of exendin-4 from a composition comprising covalently attaching it to the polypeptide.

19 CW170P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FAMCICLOVIR AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to famciclovir, as well as methods for protecting and administering famciclovir. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Famciclovir is a known phaimaceutical agent that is used in the treatment of viral infection. Its chemical name is 2-[2-(2-amino-9H-purin-9-yl)ethyl]-l,3-propanediol diacetate. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW170P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW170P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW170P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (famciclovir) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching famciclovir to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising famciclovir microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and famciclovir covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW170P

Famciclovir preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting famciclovir from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering famciclovir to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, famciclovir is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, famciclovir is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and famciclovir is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, famciclovir is CW170P

released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, famciclovir is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant^'covalentiy attached to the polypeptide and release ofthe adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching famciclovir to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, famciclovir and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW170P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize famciclovir and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of famciclovir. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Famciclovir is the subject of EP 182024 B (1991) and U.S. Patent Number 5,246,937, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises famciclovir covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture. CW170P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein, The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW170P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW170P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the canier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW170P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW170P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, famciclovir is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW170P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-famciclovir conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW170P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0 C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW170P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW170P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW170P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and famciclovir covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein famciclovir is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein famciclovir is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing famciclovir from said composition in a pH-dependent manner.

19. A method for protecting famciclovir from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of famciclovir from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching famciclovir to said polypeptide.

21. A method for delivering famciclovir to a patient comprising administering to said patient a composition comprising: a polypeptide; and famciclovir covalently attached to said polypeptide.

22. The method of claim 21 wherein famciclovir is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein famciclovir is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and famciclovir covalently attached to the polypeptide. Also provided is a method for delivery of famciclovir to a patient comprising administering to the patient a composition comprising a polypeptide and famciclovir covalently attached to the polypeptide. Also provided is a method for protecting famciclovir from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for conttolling release of famciclovir from a composition comprising covalently attaching it to the polypeptide.

19 CW171P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FAMOTIDINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to famotidine, as well as methods for protecting and administering famotidine. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Famotidine is a known pharmaceutical agent that is used in the treatment of ulcers and heartburn. It is both commercially available and readily manufactured using published sjmthetic schemes by those of ordinary skill in the art. Its stracmre is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW171P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW171P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW171P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(famotidine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching famotidine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Altematively, the present invention provides a pharmaceutical composition comprising famotidine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and famotidine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Famotidine preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW171P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. 7 he microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting famotidine from degradation comprising covalently attaching it to a polypeptide.

. The invention also provides a method for delivering famotidine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, famotidine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, famotidine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and famotidine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, famotidine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, famotidine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the CW171P

composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching famotidine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, famotidine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW171P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabϋize famotidine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of famotidine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises famotidine covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW171P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW171P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophiHcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

. Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, Jow molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW171P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW171P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW171P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these 5 polypeptides through a spacer or linker on the pendant group, which is terminated, ( preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is , distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the 10 alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- 15 dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, famotidine is covalently attached to the polypeptide via the amino groups.

The polypeptide carrier can be prepared using conventional techniques. A 20 prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) 25 and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW171P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-famotidine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product

13 CW171P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW171P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW171P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and famotidine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein famotidine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW171P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

>

17. The composition of claim 1 wherein famotidine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing famotidine from said composition in a pH-dependent manner.

19. A method for protecting famotidine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of famotidine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching famotidine to said polypeptide.

21. A method for delivering famotidine to a patient comprising administering to said patient a composition comprising: a polypeptide; and famotidine covalently attached to said polypeptide.

22. The method of claim 21 wherein famotidine is released from said composition by an enzyme-catalyzed release.

17 CW171P

23. The method of claim 21 wherein famotidine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW171P

Abstract

A composition comprising a polypeptide and famotidine covalently attached to the polypeptide. Also-provided is a method for delivery of famotidine to a patient comprising administering to the patient a composition comprising a polypeptide and famotidine covalently attached to the polypeptide. Also provided is a method for protecting famotidine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controUing release of famotidine from a composition comprising covalently attaching it to the polypeptide.

19 CW172P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FELODIPINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to felodipine, as well as methods for protecting and administering felodipine. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pha naceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Felodipine is a known phaonaceutical agent that is used in the treatment of hypertension. Its chemical name is 4-(2,3-dichlorophenyl)-l,4-dihydro-2,6-dimethyl-3,5- pyridinedicarboxylic acid ethyl methyl ester. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW172P

Active agent delivery systems are often critical for the effective delivery of a biologicaUy active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW172P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW172P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (felodipine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching felodipine to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising felodipine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and felodipine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Felodipine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW172P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting felodipine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering felodipine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, felodipine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, felodipine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and felodipine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, felodipine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, felodipine is released from the composition in a sustained release. CW172P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching felodipine to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, felodipine and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW172P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize felodipine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of felodipine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Felodipine is the subject of U.S. Patent Numbers 4,264,611 and 4,803,081, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises felodipine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary structure and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW172P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW172P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW172P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) ' 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW172P

delivery composition or over two times the maximum drug loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teπninus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutaπuc acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW172P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW172P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane fransport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-felodipine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW172P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyπolidinopyridine, dimethylaminopyridine, triethylaπύne or tributylamine.

14 CW172P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the niixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^'

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW172P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and felodipine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturaUy occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein felodipine is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW172P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein felodipine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing felodipine from said composition in a pH-dependent manner.

19. A method for protecting felodipine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of felodipine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching felodipine to said polypeptide.

21. A method for delivering felodipine to a patient comprising administering to said patient a composition comprising: a polypeptide; and felodipine covalently attached to said polypeptide.

22. The method of claim 21 wherein felodipine is released from said composition by an enzyme-catalyzed release.

17 CW172P

23. The method of claim 21 wherein felodipine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW172P

Abstract

A composition comprising a polypeptide and felodipine covalently attached to the polypeptide. Also provided is a method for delivery of felodipine to a patient comprising administering to the patient a composition comprising a polypeptide and felodipine covalently attached to the polypeptide. Also provided is a method for protecting felodipine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of felodipine from a composition comprising covalently attaching it to the polypeptide.

19 CW173P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FENOFIBRATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to fenofibrate, as well as methods for protecting and administering fenofibrate. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fenofibrate is a known phaimaceutical agent that is used in the treatment of hyperlipiemia. Its chemical name is 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid 1-methylethyl ester. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW173P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW173P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW173P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fenofibrate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching fenofibrate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising fenofibrate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fenofibrate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Fenofibrate preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW173P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fenofibrate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fenofibrate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, fenofibrate is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, fenofibrate is released in a time-dependent manner based on the pharmacokinetics of the enzjmie-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and fenofibrate is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, fenofibrate is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, fenofibrate is released from the composition in a CW173P

sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active

5 ingredients.

(

, The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching fenofibrate to a side chain of an amino acid to form an active 0 agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). 5 In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, fenofibrate and a second active agent can be copolymerized in step (c). In .another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the 0 active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a 5 carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. 0 The general applications of this invention to other active pharmaceutical agents is CW173P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize fenofibrate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fenofibrate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Fenofibrate is the subject of U.S. Patent Number 4,895,726, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises fenofibrate covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW173P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW173P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW173P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) ^' 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW173P

delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW173P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary fract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW173P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the canier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fenofibrate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW173P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can-then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW173P

Preparation of γ- Alky I Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alky I Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.^'

Preparation of Poly[γ-AIkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW173P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and fenofibrate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occuning amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein fenofibrate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW173P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein fenofibrate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fenofibrate from said composition in a pH-dependent manner.

19. A method for protecting fenofibrate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of fenofibrate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching fenofibrate to said polypeptide.

21. A method for delivering fenofibrate to a patient comprising administering to said patient a composition comprising: a polypeptide; and fenofibrate covalently attached to said polypeptide.

22. The method of claim 21 wherein fenofibrate is released from said composition by an enzyme-catalyzed release.

17 CW173P

23. The method of claim 21 wherein fenofibrate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW173P

Abstract

A composition comprising a polypeptide and fenofibrate covalently attached to the polypeptide. Also provided is a method for delivery of fenofibrate to a patient comprising administering to the patient a composition comprising a polypeptide and fenofibrate covalently attached to the polypeptide. Also provided is a method for protecting fenofibrate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of fenofibrate from a composition comprising covalently attaching it to the polypeptide.

19 CW174P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FENRETTNIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to fenretinide, as well as methods for protecting and administering fenretinide. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fenretinide is a known pharmaceutical agent that is used in the treatment of cancer. Its chemical name is N-(4-hydroxyphenyl)retinamide. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken CW174P

under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harsidy acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active, agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent CW174P

in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR CW174P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fenretinide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching fenretinide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide br polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising fenretinide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fenretinide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Fenretinide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In. a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is CW174P

an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the poljφeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fenretinide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fenretinide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, femetinide is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, femetinide is released in a time-dependent manner based on the pharmacokinetics of the enzyme.-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and fenretinide is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, fenretinide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, fenretinide is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the CW174P

composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching fenretinide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, fenretinide and a second active agent can be copoljmierized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular fransamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW174P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize fenretinide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fenretinide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention a.so allows targeted delivery of active agents to specifics sites of action.

Fenretinide is the subject of GB 1543824 (1979) - based on priority US application 628177 (1975), US 4,323,581 (1982), and US 4,665,098 (1987), , herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises fenretinide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the CW174P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW174P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW174P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW174P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terπύnus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-dmg dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

U CW174P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of acάon for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, fenretinide is covalently attached to the polypeptide via the hydroxyl.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW174P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fenretinide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW174P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution car then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW174P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW174P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and fenretinide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein fenretinide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW174P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein fenretinide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fenretinide from said composition in a pH-dependent manner.

19. A method for protecting fenretinide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of fenretinide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching fenretinide to said polypeptide.

21. A method for delivering fenretinide to a patient comprising administering to said patient a composition comprising: a polypeptide; and fenretinide covalently attached to said polypeptide.

22. The method of claim 21 wherein fenretinide is released from said composition by an enzyme-catalyzed release.

17 CW174P

23. The method of claim 21 wherein fenretinide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW174P

Abstract

A composition comprising a polypeptide and fenretinide covalentiy attached to the polypeptide. Also provided is a method for delivery of fenretinide to a patient comprising administering to the patient a composition comprising a polypeptide and fenretinide covalently attached to the polypeptide. Also provided is a method for protecting fenretinide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of fenretinide from a composition comprising covalently attaching it to the polypeptide.

19 CW175P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FENTANYL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to fentanyl, as well as methods for protecting and administering fentanyl. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fentanyl is a known pharmaceutical agent that is used in the treatment of pain. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW175P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such is prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW175P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW175P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fentanyl) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching fentanyl to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising fentanyl microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fentanyl covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Fentanyl preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW175P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fentanyl from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fentanyl to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, fentanyl is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, fentanyl is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and fentanyl is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, fentanyl is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, fentanyl is released from the composition in a sustained release. CW175P

In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching fentanyl to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, fentanyl and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether,, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is CW175P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize fentanyl and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fentanyl. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises fentanyl covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW175P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" ofthe packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW175P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogenbonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW175P

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW175P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

n CW175P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW175P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fentanyl conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW175P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can-then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably.protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyπolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW175P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the-drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ- Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the detaUs shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW175P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and fentanyl covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein fentanyl is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW175P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein fentanyl is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fentanyl from said composition in a pH-dependent manner.

19. A method for protecting fentanyl from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for confrolling release of fentanyl from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching fentanyl to said polypeptide.

21. A method for delivering fentanyl to a patient comprising administering to said patient a composition comprising: a polypeptide; and fentanyl covalently attached to said polypeptide.

22. The method of claim 21 wherein fentanyl is released from said composition by an enzyme-catalyzed release.

17 CW175P

23. The method of claim 21 wherein fentanyl is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW175P

Abstract

A composition comprising a polypeptide and fentanyl covalently attached to the polypeptide. Also provided is a method for delivery of fentanyl to a patient comprising administering to the patient a composition comprising a polypeptide and fentanyl covalently attached to the polypeptide. Also provided is a method for protecting fentanyl from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of fentanyl from a composition comprising covalently attaching it to the polypeptide.

19 CW176P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FEXOFENADINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to fexofenadine, as well as methods for protecting and administering fexofenadine. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fexofenadine is a known pharmaceutical agent that is used in the treatment of seasonal allergic rhinitis. Its chemical name is 4- [l-hydroxy-4- [4-

(hydroxydiphenylmethyl)- 1 -piperidinyl]butyl]-alpha,alpha-dimethylbenzeneacetic acid. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW176P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent deUvery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW176P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW176P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fexofenadine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching fexofenadine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising fexofenadine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fexofenadine covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturaUy occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW176P

Fexofenadine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fexofenadine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fexofenadine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, fexofenadine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, fexofenadine is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and fexofenadine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, CW176P

fexofenadine is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, fexofenadine is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching fexofenadine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, fexofenadine and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW176P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, - the invention can stabilize fexofenadine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fexofenadine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Fexofenadine is the subject of U.S. Patent Numbers 4,254,129, 5,578,610, 5,855,912, 5,932,247, and 6,037,353, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises fexofenadine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary structure and the spatial anangement of the side chains constitute the tertiary stracture. CW176P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stabUity, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW176P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophiUcity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW176P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

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molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teπninus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydjOxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW176P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Altematively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, fexofenadine is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW176P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fexofenadine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

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Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW176P

hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C.

The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW176P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

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What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and fexofenadine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more sjmthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is^' a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein fexofenadine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW176P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein fexofenadine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fexofenadine from said composition in a pH-dependent manner.

19. A method for protecting fexofenadine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of fexofenadine from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching fexofenadine to said polypeptide.

21. A method for delivering fexofenadine to a patient comprising administering to said patient a composition comprising: a polypeptide; and fexofenadine covalently attached to said polypeptide.

22. The method of claim 21 wherein fexofenadine is released from said composition by an enzyme-catalyzed release.

18 CW176P

23. The method of claim 21 wherein fexofenadine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and fexofenadine covalently attached to the polypeptide. Also provided is a method for delivery of fexofenadine to a patient comprising administering to the patient a composition comprising a polypeptide and fexofenadine covalently attached to the polypeptide. Also provided is a method for protecting fexofenadine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controUing release of fexofenadine from a composition comprising covalently attaching it to the polypeptide.

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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FILGRASTIM AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to filgrastim, as well as methods for protecting and administering filgrastim. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Filgrastim is a known pharmaceutical agent that is used in the treatment of cancer, HIV infection, pneumonia, leukopenia and skin ulcer. Its chemical name is N-L- methionyl-colony-stimulating factor (human clone 1034).

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW177P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW177P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW177P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (filgrastim) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching filgrastim to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzjmiatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising filgrastim microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and filgrastim covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Filgrastim preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide. CW177P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting filgrastim from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering filgrastim to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefened embodiment, filgrastim is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, filgrastim is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and filgrastim is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, filgrastim is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, filgrastim is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW177P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching filgrastim to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, filgrastim and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize filgrastim and prevent its digestion in the stomach. In CW177P

addition, the pharmacologic effect can be prolonged by delayed release of filgrastim. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Filgrastim is the subject of EP 237545 B (1991), based on priority application US

768959 (1985), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises filgrastim covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constimte the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW177P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW177P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level ofconformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. GeneraUy, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW177P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW177P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW177P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, filgrastim is covalently attached to the polypeptide via a peptide bond.

The polypeptide canier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW177P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-filgrastim conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AciόVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to

0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW177P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinop τidine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW177P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW177P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and filgrastim covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein filgrastim is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW177P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein filgrastim is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing filgrastim from said composition in a pH-dependent manner.

19. A method for protecting filgrastim from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of filgrastim from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching filgrastim to said polypeptide.

21. A method for delivering filgrastim to a patient comprising administering to said patient a composition comprising: a polypeptide; and filgrastim covalently attached to said polypeptide.

22. The method of claim 21 wherein filgrastim is released from said composition by an enzyme-catalyzed release.

17 CW177P

23. The method of claim 21 wherein filgrastim is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW177P

Abstract

A composition comprising a polypeptide .and filgrastim covalently attached to the polypeptide. Also provided is a method for delivery of filgrastim to a patient comprising administering to the patient a composition comprising a polypeptide and filgrastim covalently attached to the polypeptide. Also provided is a method for protecting filgrastim from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of filgrastim from a composition comprising covalently attaching it to the polypeptide.

19 CW178P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FINASTERIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to finasteride, as well as methods for protecting and administering finasteride. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Finasteride is a known phaimaceutical agent that is used in the treatment of cancer, benign prostate hypertrophy, alopecia and acne. Its chemical name is

(5alpha,17beta)-N-(l,l-dimethylethyl)-3-oxo-4-azaandrost-l-ene-17-carb oxamide. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical CW178P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabUity of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW178P

shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW178P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labUe active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (finasteride) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching finasteride to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising finasteride microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and finasteride covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW178P

Finasteride preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- teiminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting finasteride from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering finasteride to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, finasteride is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, finasteride is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and finasteride is released from the composition by dissolution of the πucroencapsulating agent. In another prefened embodiment, finasteride is released CW178P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, finasteride is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of: (a) attaching finasteride to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, finasteride and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW178P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize finasteride and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of finasteride. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Finasteride is the subject of U.S. Patent Numbers 5,377,584, 4,760,071, 5,547,957, 5,571,817 and 5,886,184, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises finasteride covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding CW178P

are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW178P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophiUcity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW178P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a poljφeptide is considered. For example, a decamer.of glycine (MW=588) linked to aspirin would have a total molecular

10 CW178P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be -rted into the gamma ester of glutamic acid N-

u CW178P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain ofthe polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, finasteride is covalently attached to the polypeptide via an amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW178P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-finasteride conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW178P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can- then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW178P

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW178P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and finasteride covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein finasteride is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW178P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein finasteride is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing finasteride from said composition in a pH-dependent manner.

19. A method for protecting finasteride from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of finasteride from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching finasteride to said polypeptide.

21. A method for delivering finasteride to a patient comprising administering to said patient a composition comprising: a polypeptide; and finasteride covalently attached to said polypeptide.

22. The method of claim 21 wherein finasteride is released from said composition by an enzyme-catalyzed release.

17 CW178P

23. The method of claim 21 wherein finasteride is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW178P

Abstract

A composition comprising a polypeptide and finasteride covalently attached to the polypeptide. Also provided is a method for delivery of finasteride to a patient comprising administering to the patient a composition comprising a polypeptide and finasteride covalently attached to the polypeptide. Also provided is a method for protecting finasteride from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of finasteride from a composition comprising covalently attaching it to the polypeptide.

19 CW179P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FLECAINIDE ACETATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to flecainide acetate, as weU as methods for protecting and administering flecainide acetate. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Flecainide acetate is a known pharmaceutical agent that is used in the treatment of arrythmia. Its chemical name is N-(2-piperidinylmethyl)-2,5-bis(2,2,2- ttifluoroethoxy)benzamide. Its stracture is:

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW179P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW179P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW179P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane ofthe intestines is Umited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (flecainide acetate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching flecainide acetate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising flecainide acetate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and flecainide acetate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Flecainide acetate preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW179P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting flecainide acetate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering flecainide acetate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, flecainide acetate is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, flecainide acetate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and flecainide acetate is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, flecainide acetate is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, flecainide acetate is CW179P

released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching flecainide acetate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, flecainide acetate and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW179P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize flecainide acetate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of flecainide acetate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Flecainide acetate is the subject of U.S. Patent Number 4,642,384, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises flecainide acetate covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW179P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining- forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW179P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophiUcity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophϋicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW179P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW179P

delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drug conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

u CW179P

any suitable initiator as shown in Fig.4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart 5 specific properties to the drag delivery system.

(

I The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not Hmited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these

10 polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the

15 alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- 20 dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, flecainide acetate is covalentiy attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A 25 prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW179P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficientiy via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-flecainide acetate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW179P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can- then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW179P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be Hmited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW179P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and flecainide acetate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein flecainide acetate is covalently attached to a side chain, the N-terminus or the C-terminus of said poljφeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW179P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein flecainide acetate is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing flecainide acetate from said composition in a pH-dependent manner.

19. A method for protecting flecainide acetate from degradation comprising covalently attaching said active agent to a polypeptide. *

20. A method for controlling release of flecainide acetate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching flecainide acetate to said polypeptide.

21. A method for delivering flecainide acetate to a patient comprising administering to said patient a composition comprising: a polypeptide; and flecainide acetate covalently attached to said polypeptide.

22. The method of claim 21 wherein flecainide acetate is released from said composition by an enzyme-catalyzed release.

17 CW179P

23. The method of claim 21 wherein flecainide acetate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW179P

Abstract

A composition comprising a polypeptide and flecainide acetate covalentiy attached to the polypeptide. Also provided is a method for delivery of flecainide acetate to a patient comprising administering to the patient a composition comprising a polypeptide and flecainide acetate covalently attached to the polypeptide. Also provided is a method for protecting flecainide acetate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of flecainide acetate from a composition comprising covalently attaching it to the polypeptide.

19 CW180P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FLUCONAZOLE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to fluconazole, as well as methods for protecting and administering fluconazole. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fluconazole is a known pharmaceutical agent that is used in the treatment of fungal infections. Its chemical name is alpha-(2,4-difluorophenyl)-alpha-(lH- 1,2,4- triazol- 1 -ylmethyl)- 1 H- 1 ,2,4-triazole- 1 -ethanol. Its stracmre is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW180P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW180P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW180P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fluconazole) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching fluconazole to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising fluconazole microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fluconazole covalently attached to the polypeptide. Preferably, the polypeptide is (i).an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (Hi) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Fluconazole preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW180P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceuticaUy acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fluconazole from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fluconazole to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, fluconazole is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, fluconazole is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and fluconazole is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, fluconazole is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, fluconazole is released from the CW180P

composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching fluconazole to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, fluconazole and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an ariiino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW180P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize fluconazole and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fluconazole. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Fluconazole is the subject of U.S. Patent Numbers 4,404,216 and 4,416,682, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises fluconazole covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturaUy occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW180P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW180P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired; For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW180P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the Ueum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B^ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW180P

delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchlorofoπnate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW180P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, fluconazole is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW180P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fluconazole conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW180P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW180P

Preparation of γ-Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW180P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and fluconazole covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein fluconazole is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW180P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

5 14. The composition of claim 1 wherein said composition is in the form of an

' ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an 10 oral suspension.

17. The composition of claim 1 wherein fluconazole is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fluconazole from said composition in a pH-dependent manner.

15 19. A method for protecting fluconazole from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of fluconazole from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching fluconazole to said polypeptide.

20 21. A method for delivering fluconazole to a patient comprising administering to said patient a composition comprising: a polypeptide; and fluconazole covalently attached to said polypeptide.

22. The method of claim 21 wherein fluconazole is released from said 25 composition by an enzyme-catalyzed release.

17 CW180P

23. The method of claim 21 wherein fluconazole is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW180P

Abstract

A composition comprising a polypeptide and fluconazole covalentiy attached to the polypeptide. Also provided is a method for delivery of fluconazole to a patient comprising administering to the patient a composition comprising a polypeptide and fluconazole covalently attached to the polypeptide. Also provided is a method for protecting fluconazole from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of fluconazole from a composition comprising covalentiy attaching it to the poljφeptide.

19 CW181P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FLUDROCORTISONE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to fludrocortisone, as well as methods for protecting and administering fludrocortisone. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fludrocortisone is a known pharmaceutical agent that is used in the treatment of epilepsy. Its chemical name is 5,5-diphenyl-3-[(phosphonooxy)methyl]-2,4- imidazolidinedion. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW181P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW181P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW181P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fludrocortisone) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching fludrocortisone to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising fludrocortisone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fludrocortisone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one ofthe twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Fludrocortisone preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW181P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fludrocortisone from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fludrocortisone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, fludrocortisone is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, fludrocortisone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and fludrocortisone is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, fludrocortisone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, fludrocortisone is CW181P

released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching fludrocortisone to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, fludrocortisone and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW181P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize fludrocortisone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fludrocortisone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Fludrocortisone is the subject of U.S. Patent Number 4,260,769 (1981), and EP

473687 B (1996), based on priority application US 356948 (1989), herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises fludrocortisone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW181P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW181P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the poljφeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW181P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW181P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Altematively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW181P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine.. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not Hmited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, fludrocortisone is covalently attached to the polypeptide via the phosphate group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW181P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fludrocortisone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW181P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can-then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product r precipitated out in ether and purified using gel permeation chromatography (GPC) or

I dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

5 In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product 0 is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include 5 dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW181P

Preparation of γ-Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW181P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and fludrocortisone covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein fludrocortisone is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW181P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein fludrocortisone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fludrocortisone from said composition in a pH-dependent manner.

19. A method for protecting fludrocortisone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of fludrocortisone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching fludrocortisone to said polypeptide.

21. A method for delivering fludrocortisone to a patient comprising administering to said patient a composition comprising: a polypeptide; and fludrocortisone covalently attached to said polypeptide.

22. The method of claim 21 wherein fludrocortisone is released from said composition by an enzyme-catalyzed release.

17 CW181P

23. The method of claim 21 wherein fludrocortisone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalentiy attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW181P

Abstract

A composition comprising a polypeptide and fludrocortisone covalently attached to the polypeptide. Also provided is a method for delivery of fludrocortisone to a patient comprising administering to the patient a composition comprising a polypeptide and fludrocortisone covalently attached to the polypeptide. Also provided is a method for protecting fludrocortisone from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of fludrocortisone from a composition comprising covalently attaching it to the polypeptide.

19 CW182P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FLUMAZENIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to flumazenil, as well as methods for protecting and administering flumazenil. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Flumazenil is a known phaimaceutical agent that is used in the treatment of depression and liver disease. Its chemical name is 8-fluoro-5,6-dihydro-5-methyl-6-oxo- 4H-imidazo[l,5-a][l,4]benzodiazepine-3-carboxylic acid ethyl ester. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW182P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeabiUty of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffiision into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW182P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable^" or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW182P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (flumazenil) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching flumazenil to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising flumazenil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and flumazenil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Flumazenil preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW182P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terπύnus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting flumazenil from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering flumazenil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, flumazenil is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, flumazenil is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and flumazenil is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, flumazenil is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, flumazenil is released from the composition in a sustained CW182P

release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching flumazenil to a side chain of an amino acid to form an active agent/amino acid complex; (b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, flumazenil and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW182P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize flumazenil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of flumazenil. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced'. The invention also allows targeted delivery of active agents to specifics sites of action.

Flumazenil is the subject of U.S. Patent Number 4,316,839, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises flumazenil covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW182P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and - at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW182P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this nvention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW182P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW182P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW182P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutaπuc acid-drug dimer. This carrier peptide-drug conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW182P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-flumazenil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW182P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole foUowed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW182P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the' drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW182P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and flumazenil covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said poljφeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropoljmier of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein flumazenil is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW182P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein flumazenil is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing flumazenil from said composition in a pH-dependent manner.

19. A method for protecting flumazenil from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of flumazenil from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching flumazenil to said polypeptide.

21. A method for delivering flumazenil to a patient comprising administering to said patient a composition comprising: a polypeptide; and flumazenil covalently attached to said polypeptide.

22. The method of claim 21 wherein flumazenil is released from said composition by an enzyme-catalyzed release.

17 CW182P

23. The method of claim 21 wherein flumazenil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW182P

Abstract

A composition comprising a polypeptide and flumazenil covalentiy attached to the polypeptide. Also provided is a method for delivery of flumazenil to a patient comprising administering to the patient a composition comprising a polypeptide and flumazenil covalently attached to the polypeptide. Also provided is a method for protecting flumazenil from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of flumazenil from a composition comprising covalently attaching it to the polypeptide.

19 CW183P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FLUOXETINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to fluoxetine, as well as methods for protecting and administering fluoxetine. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fluoxetine is a known pharmaceutical agent that is used in the treatment of depression. Its chemical name is (N-methyl 3-(p-trifluorornethylphenoxy)-3- phenylpropylamine. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW183P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW183P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in tum, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW183P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fluoxetine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching fluoxetine to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising fluoxetine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fluoxetine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Fluoxetine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW183P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terπύnus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fluoxetine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fluoxetine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, fluoxetine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, fluoxetine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and fluoxetine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, fluoxetine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, fluoxetine is released from the composition in a sustained release. CW183P

In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a poljφeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching fluoxetine to a side chain of an amino acid to form an active agent/amino acid complex; (b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second . agent, fluoxetine and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW183P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize fluoxetine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fluoxetine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Fluoxetine is the subject of U.S. Patent Number 4,329,356, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises fluoxetine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW183P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW183P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW183P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW183P

delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-teoninus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular ttansamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW183P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teiminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, fluoxetine is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW183P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fluoxetine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW183P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can^* then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW183P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the^" drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alk l Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW183P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and fluoxetine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein fluoxetine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW183P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

₍5 14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an 10 oral suspension.

17. The composition of claim 1 wherein fluoxetine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fluoxetine from said composition in a pH-dependent manner.

15 19. A method for protecting fluoxetine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of fluoxetine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching fluoxetine to said polypeptide.

20 21. A method for delivering fluoxetine to a patient comprising administering to said patient a composition comprising: a polypeptide; and fluoxetine covalently attached to said polypeptide.

22. The method of claim 21 wherein fluoxetine is released from said composition 25 by an enzyme-catalyzed release.

17 CW183P

23. The method of claim 21 wherein fluoxetine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW183P

Abstract

A composition comprising a polypeptide and fluoxetine covalently attached to the polypeptide. Also provided is a method for delivery of fluoxetine to a patient comprising administering to the patient a composition comprising a polypeptide and fluoxetine covalently attached to the polypeptide. Also provided is a method for protecting fluoxetine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of fluoxetine from a composition comprising covalentiy attaching it to the polypeptide.

19 CW184P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FLUTAMIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to flutamide, as well as methods for protecting and administering flutamide. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Flutamide is a known pharmaceutical agent that is used in the treatment of prostate cancer. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant,, or an inhibitor. CW184P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW184P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW184P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (flutamide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching flutamide to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising flutamide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and flutamide covalently attached to the polypeptide. Preferably, the polypeptide is (i)_. an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Flutamide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW184P

carboxylic acid and is covalently attached to the N-terminus of the poljφeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting flutamide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering flutamide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, flutamide is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, flutamide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and flutamide is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, flutamide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, flutamide is released from the composition in a sustained release. CW184P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching flutamide to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, flutamide and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is CW184P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize flutamide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of flutamide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal fract can be enhanced. The invention also aUows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises flutamide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and poljφeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW184P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major force's contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW184P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given appUcation. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW184P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controUed.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin e (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW184P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW184P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acylttiazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, flutamide is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW184P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-flutamide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW184P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW184P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture re fluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^•

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW184P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and flutamide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein flutamide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW184P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein flutamide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing flutamide from said composition in a pH-dependent manner.

19. A method for protecting flutaπude from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of flutamide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching flutamide to said polypeptide.

21. A method for delivering flutamide to a patient comprising administering to said patient a composition comprising: a polypeptide; and flutamide covalently attached to said polypeptide.

22. The method of claim 21 wherein flutamide is released from said composition by an enzyme-catalyzed release.

17 CW184P

23. The method of claim 21 wherein flutamide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW184P

Abstract

A composition comprising a polypeptide and flutamide covalently attached to the polypeptide. Also provided is a method for delivery of flutamide to a patient comprising administering to the patient a composition comprising a polypeptide and flutaπude covalently attached to the polypeptide. Also provided is a method for protecting flutamide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for conttolling release of flutamide from a composition comprising covalently attaching it to the polypeptide.

19 CW185P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FLUVASTATIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to fluvastatin, as well as methods for protecting and administering fluvastatin. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fluvastatin is a known pharmaceutical agent that is used in the treatment of hyperlipidemia. Its chemical name is (3R,5S,6E)-rel-7-[3-(4-fluorophenyl)-l-(l- methylethyl)-lH-indol-2-yl]-3,5-dihydroxy-6-heptenoic acid. Its structure is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW185P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW185P

shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW185P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fluvastatin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching fluvastatin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising fluvastatin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fluvastatin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW185P

Fluvastatin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fluvastatin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fluvastatin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, fluvastatin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, fluvastatin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and fluvastatin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, fluvastatin is released CW185P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, fluvastatin is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching fluvastatin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, fluvastatin and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW185P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize fluvastatin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fluvastatin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Fluvastatin is the subject of U.S. Patent Number 5,354,772, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises fluvastatin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW185P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW185P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW185P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW185P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could 5 conceivably have a loading of 58%, although this may not be entirely practical.

( The alcohol, amine or carboxylic acid group of an active agent may be covalentiy

( attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N-

10 terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the

15 preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the

20 peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid

25 moiety serves as a spacer between the peptide and the drag as shown in Fig.4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively,

30 the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N-

U CW185P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not Umited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, fluvastatin is covalentiy attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW185P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fluvastatin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW185P

AciάVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can_, then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

—

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine. .

14 CW185P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW185P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein fluvastatin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fluvastatin from said composition in a pH-dependent manner.

19. A method for protecting fluvastatin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of fluvastatin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching fluvastatin to said polypeptide.

21. A method for delivering fluvastatin to a patient comprising administering to said patient a composition comprising: a polypeptide; and fluvastatin covalently attached to said polypeptide.

22. The method of claim 21 wherein fluvastatin is released from said composition by an enzyme-catalyzed release.

17 CW185P

23. The method of claim 21 wherein fluvastatin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW185P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and fluvastatin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein fluvastatin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW185P

Abstract

A composition comprising a polypeptide and fluvastatin covalently attached to the polypeptide. Also provided is a method for delivery of fluvastatin to a patient comprising administering to the patient a composition comprising a polypeptide and fluvastatin covalently attached to the polypeptide. Also provided is a method for protecting fluvastatin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of fluvastatin from a composition comprising covalently attaching it to the polypeptide.

19 CW186P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FLUVOXAMINE MALEATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to fluvoxamine maleate, as well as methods for protecting and administering fluvoxamine maleate. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fluvoxamine maleate is a known pharmaceutical agent that is used in the treatment of depression and anxiety. Its chemical name is 5-methoxy-l-[4-

(trifluoromethyl)phenyl]-l-pentanone (E)-O-(2-aminoethyl)oxime. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor. CW186P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and fransport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW186P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in. CW186P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fluvoxamine maleate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching fluvoxamine maleate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising fluvoxamine maleate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fluvoxamine maleate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Fluvoxamine maleate preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active CW186P

, agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fluvoxamine maleate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fluvoxamine maleate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, fluvoxamine maleate is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, fluvoxamine maleate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and fluvoxamine maleate is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, fluvoxamine maleate is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, fluvoxamine CW186P

maleate is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching fluvoxamine maleate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, fluvoxamine maleate and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW186P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize fluvoxamine maleate and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fluvoxamine maleate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Fluvoxamine maleate is the subject of GB1535226 (1978), herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises fluvoxamine maleate covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW186P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW186P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophϋicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW186P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon aid drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be confroUed.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW186P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW186P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, fluvoxamine maleate is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW186P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fluvoxamine maleate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW186P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW186P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the-drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW186P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and fluvoxamine maleate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein fluvoxamine maleate is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW186P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is iri_: the form of an oral suspension.

17. The composition of claim 1 wherein fluvoxamine maleate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fluvoxamine maleate from said composition in a pH-dependent manner.

19. A method for protecting fluvoxamine maleate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of fluvoxamine maleate from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching fluvoxamine maleate to said polypeptide.

21. A method for delivering fluvoxamine maleate to a patient comprising administering to said patient a composition comprising: a polypeptide; and fluvoxamine maleate covalently attached to said polypeptide.

22. The method of claim 21 wherein fluvoxamine maleate is released from said composition by an enzyme-catalyzed release.

17 CW186P

23. The method of claim 21 wherein fluvoxamine maleate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW186P

Abstract

A composition comprising a polypeptide and fluvoxamine maleate covalently attached to the polypeptide. Also provided is a method for delivery of fluvoxamine maleate to a patient comprising administering to the patient a composition comprising a polypeptide and fluvoxamine maleate covalently attached to the polypeptide. Also provided is a method for protecting fluvoxamine maleate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of fluvoxamine maleate from a composition comprising covalently attaching it to the polypeptide.

19 CW187P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FOLLITROPIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to follitropin, as well as methods for protecting and administering follitropin. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Follitropin is a known pharmaceutical agent that is used in the treatment of infertility. Its chemical name is follicle-stimulating hormone (human alpha-subunit reduced), complex with follicle-stimulating hormone (human beta-subunit reduced).

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW187P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an oraUy administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW187P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, usemas been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW187P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (follitropin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching follitropin to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising follitropin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and follitropin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Follitropin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide. CW187P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salf. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting follitropin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering follitropin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a preferred embodiment, follitropin is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, follitropin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and follitropin is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, follitropin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, follitropin is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW187P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching follitropin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, follitropin and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize follitropin and prevent its digestion in the stomach. In CW187P

addition, the pharmacologic effect can be prolonged by delayed release of follitropin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Follitropin is the subject of WO 95/19991 (1995) and U.S. Patent Numbers

4,589,402, 5,270,057, and 5,767,251, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises follitropin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stabiUty is the solid reference state. CW187P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic ^"consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW187P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino.acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level ofconformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW187P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW187P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW187P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, follitropin is covalently attached to the polypeptide via a peptide bond.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW187P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-foUitropin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product

13 CW187P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus ofthe peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW187P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- lkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW187P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and follitropin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein follitropin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW187P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein follitropin is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing follitropin from said composition in a pH-dependent manner.

19. A method for protecting follitropin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of follitropin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching follitropin to said polypeptide.

21. A method for delivering follitropin to a patient comprising administering to said patient a composition comprising: a polypeptide; and follitropin covalently attached to said polypeptide.

22. The method of claim 21 wherein follitropin is released from said composition by an enzyme-catalyzed release.

17 CW187P

23. The method of claim 21 wherein follitropin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW187P

Abstract

A composition comprising a polypeptide and follitropin covalentiy attached to the polypeptide. Also provided is a method for delivery of follitropin to a patient comprising administering to the patient a composition comprising a polypeptide and follitropin covalently attached to the polypeptide. Also provided is a method for protecting follitropin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of follitropin from a composition comprising covalently attaching it to the polypeptide.

19 CW188P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FORMOTEROL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to formoterol, as well as methods for protecting and administering formoterol. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Formoterol is a known pharmaceutical agent that is used in the treatment of asthma. Its chemical name is rel-N-[2-hydroxy-5-[(lR)-l-hydroxy-2-[[(lR)-2-(4- methoxypheny)-l-methylethyl]amino]ethyl]phenyl]formamide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW188P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW188P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW188P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absorption through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (formoterol) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching formoterol to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising formoterol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and formoterol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Formoterol preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW188P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- te ninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting formoterol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering formoterol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, foonoterol is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, foonoterol is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and formoterol is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, foonoterol is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, foonoterol is released from the composition in a sustained CW188P

release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching formoterol to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, formoterol and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW188P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize formoterol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of formoterol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Formoterol is the subject of GB 1415256 (1975), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises formoterol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW188P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining^* forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW188P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polj eptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW188P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW188P

delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW188P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysiπe, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release ofthe drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, formoterol is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Altematively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW188P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-formoterol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW188P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can. then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiiπύde or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW188P

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the-drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl GIutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW188P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and formoterol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein formoterol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW188P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein formoterol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing formoterol from said composition in a pH-dependent manner.

19. A method for protecting formoterol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controUing release of formoterol from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching formoterol to said polypeptide.

21. A method for delivering formoterol to a patient comprising administering to said patient a composition comprising: a polypeptide; and formoterol covalently attached to said polypeptide.

22. The method of claim 21 wherein formoterol is released from said composition by an enzyme-catalyzed release.

17 CW188P

23. The method of claim 21 wherein formoterol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW188P

Abstract

A composition comprising a polypeptide and formoterol covalently attached to the polypeptide. Also provided is a method for delivery of formoterol to a patient comprising administering to the patient a composition comprising a polypeptide and formoterol covalently attached to the polypeptide. Also provided is a method for protecting formoterol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of formoterol from a composition comprising covalently attaching it to the polypeptide.

19 CW189P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FOSINOPRIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to fosinopril, as well as methods for protecting and administering fosinopril. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness ofthe pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fosinopril is a known phaimaceutical agent that is used in the treatment of hypertension. Its chemical name is [l[S*(R*)],2alpha.,4beta]-4-cyclohexyl-l-[[[2- methyl-l-(l-oxopropoxy)propoxy](4-phenylbutyl)phosphinyl]acetyl]-L-proline. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW189P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW189P

shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in tum, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW189P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fosinopril) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching fosinopril to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising fosinopril microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fosinopril covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW189P

Fosinopril preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fosinopril from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fosinopril to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, fosinopril is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, fosinopril is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and fosinopril is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, fosinopril is released CW189P

from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, fosinopril is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching fosinopril to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, fosinopril and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutaπuc acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW189P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize fosinopril and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fosinopril. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Fosinopril is the subject of U.S. Patent Number 4,337,201, 4,384,123, and 5,006,344, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises fosinopril covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (U) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracmre and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW189P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW189P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will . ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW189P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

VaUne 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer.of glycine (MW=588) linked to aspirin would have a total molecular

10 CW189P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to rjoly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW189P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, fosinopril is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Altematively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW189P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fosinopril conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW189P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW189P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW189P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and fosinopril covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein fosinopril is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW189P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein fosinopril is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fosinopril from said composition in a pH-dependent manner.

19. A method for protecting fosinopril from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of fosinopril from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching fosinopril to said polypeptide.

21. A method for delivering fosinopril to a patient comprising administering to said patient a composition comprising: a polypeptide; and fosinopril covalently attached to said polypeptide.

22. The method of claim 21 wherein fosinopril is released from said composition by an enzyme-catalyzed release.

17 CW189P

23. The method of claim 21 wherein fosinopril is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW189P

Abstract

A composition comprising a polypeptide and fosinopril covalently attached to the polypeptide. Also proyided is a method for delivery of fosinopril to a patient comprising administering to the patient a composition comprising a polypeptide and fosinopril covalently attached to the polypeptide. Also provided is a method for protecting fosinopril from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for conttolling release of fosinopril from a composition comprising covalently attaching it to the polypeptide.

19 CW190P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FOSPHENYTOIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to fosphenytoin, as well as methods for protecting and administering fosphenytoin. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Fosphenytoin is a known phaonaceutical agent that is used in the treatment of epilepsy. Its chemical name is 5,5-diphenyl-3-[(phosphonooxy)methyl]-2,4- imidazolidinedione. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW190P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bUe acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW190P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW190P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absorption through the bmsh-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (fosphenytoin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching fosphenytoin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oUgopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzjmiatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising fosphenytoin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and fosphenytoin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Fosphenytoin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW190P

carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus ofthe polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting fosphenytoin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering fosphenytoin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, fosphenytoin is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, fosphenytoin is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and fosphenytoin is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, fosphenytoin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, fosphenytoin is released from the CW190P

composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising u polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching fosphenytoin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, fosphenytoin and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW190P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize fosphenytoin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of fosphenytoin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Fosphenytoin is the subject of U.S. Patent Numbers 4,260,769 and 4,925,860, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises fosphenytoin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (u) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW190P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the soUd reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW190P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophihcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given appUcation. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW190P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophUic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) ^• 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW190P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain ofthe oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. this intermediate can then be polymerized, as described above, using

1 1 CW190P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, fosphenytoin is covalentiy attached to the polypeptide via the phosphate or amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW190P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-fosphenytoin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

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Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0 C. The solution cai then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include p Ηolidinop ridine, dimethylaminopyridine, triethylamine or tributylamine.

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Preparation of γ-AIkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the- drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. '

Preparation of Poly [γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

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What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and fosphenytoin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein fosphenytoin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein fosphenytoin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing fosphenytoin from said composition in a pH-dependent manner.

19. A method for protecting fosphenytoin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of fosphenytoin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching fosphenytoin to said polypeptide.

21. A method for delivering fosphenytoin to a patient comprising administering to said patient a composition comprising: a polypeptide; and fosphenytoin covalently attached to said polypeptide.

22. The method of claim 21 wherein fosphenytoin is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein fosphenytoin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

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Abstract

A composition comprising a polypeptide and fosphenytoin covalently attached to the polypeptide. Also provided is a method for delivery of fosphenytoin to a patient comprising administering to the patient a composition comprising a polypeptide and fosphenytoin covalently attached to the polypeptide. Also provided is a method for protecting fosphenytoin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for confrolling release of fosphenytoin from a composition comprising covalently attaching it to the polypeptide.

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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING FUROSEMIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to furosemide, as well as methods for protecting and administering furosemide. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Furosemide is a known pharmaceutical agent that is used in the treatment of edema and hypertension. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW191P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzjmie degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW191P

shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW191P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (furosemide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching furosemide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising furosemide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and furosemide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW191P

Furosemide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and "is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting furosemide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering furosemide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, furosemide is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, furosemide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and furosemide is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, furosemide is CW191P

released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, furosemide is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching furosemide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

^" (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, furosemide and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW191P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize furosemide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of furosemide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises furosemide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (U) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropoljmier of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW191P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded . protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW191P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW191P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B_ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW191P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Altematively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

u CW191P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, furosemide is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW191P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-furoseπύde conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW191P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW191P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture re fluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW191P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and furosemide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more sjmthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is' a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein furosemide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW191P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

5 , 14. The composition of claim 1 wherein said composition is in the form of an

₍ ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein furosemide is conformationaUy protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing furosemide from said composition in a pH-dependent manner.

19. A method for protecting furosemide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of furosemide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching furosemide to said polypeptide.

0 21. A method for delivering furosemide to a patient comprising administering to said patient a composition comprising: a polypeptide; and furosemide covalently attached to said polypeptide.

22. The method of claim 21 wherein furosemide is released from said 5 composition by an enzyme-catalyzed release.

17 CW191P

23. The method of claim 21 wherein furosemide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and furosemide covalentiy attached to the polypeptide. Also provided is a method for delivery of furosemide to a patient comprising administering to the patient a composition comprising a polypeptide and furosemide covalently attached to the polypeptide. Also provided is a method for protecting furosemide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of furosemide from a composition comprising covalently attaching it to the polypeptide.

18 CW192P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GABAPENTTN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to gabapentin, as well as methods for protecting and administering gabapentin. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Gabapentin is a known pharmaceutical agent that is used in the treatment of epilepsy and depression. Its chemical name is l-(aminomethyl)cyclohexaneacetic acid. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken CW192P

under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedTy provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent CW192P

in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system here the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight πucrospheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR CW192P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (gabapentin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching gabapentin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising gabapentin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and gabapentin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Gabapentin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is CW192P

an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting gabapentin from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering gabapentin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, gabapentin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, gabapentin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and gabapentin is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, gabapentin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, gabapentin is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be CW192P

microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching gabapentin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, gabapentin and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW192P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabUize gabapentin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of gabapentin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Gabapentin is the subject of U.S. Patent Numbers 4,087,544; 4,894,476, 5,084,479, and 6,054,482, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises gabapentin covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the poljφeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the CW192P

protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW192P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW192P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW192P

delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain ofthe oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW192P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, gabapentin is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW192P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the canier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the ttansported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-gabapentin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW192P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0 C. The solution can-then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyπolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW192P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the^* drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW192P

What is claimed is:

L A pharmaceutical composition comprising: a polypeptide; and gabapentin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein gabapentin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW192P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

5c 14. The composition of claim 1 wherein said composition is in the form of an

, ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an 0 oral suspension.

17. The composition of claim 1 wherein gabapentin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ^' gabapentin from said composition in a pH-dependent manner.

5 19. A method for protecting gabapentin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of gabapentin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching gabapentin to said polypeptide.

0 21. A method for delivering gabapentin to a patient comprising administering to said patient a composition comprising: a polypeptide; and gabapentin covalently attached to said polypeptide.

22. The method of claim 21 wherein gabapentin is released from said 5 composition by an enzyme-catalyzed release.

17 CW192P

23. The method of claim 21 wherein gabapentin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW192P

Abstract

A composition comprising a polypeptide and gabapentin covalently attached to the polypeptide. Also provided is a method for delivery of gabapentin to a patient comprising administering to the patient a composition comprising a polypeptide and gabapentin covalently attached to the polypeptide. Also provided is a method for protecting gabapentin from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of gabapentin from a composition comprising covalently attaching it to the polypeptide.

19 CW193P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GADODIAMIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to gadodiamide, as well as methods for protecting and administering gadodiamide. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Gadodiamide is a known pharmaceutical agent that is used as a diagnostic agent in MRL Its chemical name is [5,8-bis(carboxymethyl)-l l-[2-(methylamino)-2-oxoethyl]- 3-oxo- 2,5,8,1 l-tetraazattidecan-13-oato(3-)]gadolinium. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW193P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW193P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW193P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(gadodiamide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching gadodiamide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising gadodiamide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and gadodiamide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Gadodiamide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW193P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting gadodiamide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering gadodiamide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, gadodiamide is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, gadodiamide is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and gadodiamide is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, gadodiamide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, gadodiamide is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and CW193P

release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a 5 polypeptide and an active agent covalently attached to the polypeptide. The method ' comprises the steps of:

' (a) attaching gadodiamide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) 10 from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second

IS agent, gadodiamide and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an

20 amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an

25 amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, 30 incoφorated herein by reference. CW193P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize gadodiamide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of gadodiamide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Gadodiamide is the subject of U.S. Patent Number 4,687,659, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises gadodiamide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW193P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW193P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophϋicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW193P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW193P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutaπuc acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW193P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, gadodiamide is covalently attached to the polypeptide via the amino or hydroxy group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW193P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-gadodiamide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW193P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW193P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the-drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW193P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and gadodiamide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein gadodiamide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW193P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein gadodiamide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing gadodiamide from said composition in a pH-dependent manner.

19. A method for protecting gadodiamide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of gadodiamide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching gadodiamide to said polypeptide.

21. A method for delivering gadodiamide to a patient comprising administering to said patient a composition comprising: a polypeptide; and gadodiamide covalently attached to said polypeptide.

22. The method of claim 21 wherein gadodiamide is released from said composition by an enzyme-catalyzed release.

17 CW193P

23. The method of claim 21 wherein gadodiamide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW193P

Abstract

A composition comprising a polypeptide and gadodiamide covalently attached to the polypeptide. Also provided is a method for delivery of gadodiamide to a patient comprising administering to the patient a composition comprising a polypeptide and gadodiamide covalently attached to the polypeptide. Also provided is a method for protecting gadodiamide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of gadodiamide from a composition comprising covalently attaching it to the polypeptide.

19 CW194P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GADOPENTETATE DIMEGLUMINE AND METHODS OF MAKING AND

USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to gadopentetate dimeglumine, as well as methods for protecting and administering gadopentetate dimeglumine. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Gadopentetate dimeglumine is a known pharmaceutical agent that is used for imaging in brain scans.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW194P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an oraUy administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW194P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW194P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (gadopentetate dimeglumine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching gadopentetate dimeglumine to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Altematively, the present invention provides a pharmaceutical composition comprising gadopentetate dimeglumine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and gadopentetate dimeglumine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Gadopentetate dimeglumine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet CW194P

another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting gadopentetate dimeglumine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering gadopentetate dimeglumine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, gadopentetate dimegluπune is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, gadopentetate dimeglumine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and gadopentetate dimeglumine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, gadopentetate dimegluπune is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, gadopentetate dimeglumine is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The CW194P

adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching gadopentetate dimeglumine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, gadopentetate dimeglumine and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW194P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabUize gadopentetate dimeglumine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of gadopentetate dimeglumine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises gadopentetate dimeglumine covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iU) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic aπuno acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW194P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW194P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW194P

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW194P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or

5 polypeptide. The location of attachment depends somewhat on the functional group

( selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N-

< terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples,

10 the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate

15 with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier.

20 Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will

25 then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is

30 polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW194P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not Umited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-dfug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW194P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-gadopentetate dimeglumine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be

13 CW194P

stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutaπuc acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW194P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-AIkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount Of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW194P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and gadopentetate dimeglumine covalently attached to sajd polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropoljmier of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein gadopentetate dimeglumine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW194P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein gadopentetate dimeglumine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing gadopentetate dimeglumine from said composition in a pH-dependent manner.

19. A method for protecting gadopentetate dimeglumine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of gadopentetate dimeglumine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching gadopentetate dimeglumine to said polypeptide.

21. A method for delivering gadopentetate dimeglumine to a patient comprising administering to said patient a composition comprising: a polypeptide; and gadopentetate dimeglumine covalently attached to said polypeptide.

22. The method of claim 21 wherein gadopentetate dimeglumine is released from said composition by an enzyme-catalyzed release.

17 CW194P

23. The method of claim 21 wherein gadopentetate dimeglumine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW194P

Abstract

A composition comprising a polypeptide and gadopentetate dimeglumine covalently attached to the polypeptide. Also provided is a method for delivery of gadopentetate dimeglumine to a patient comprising administering to the patient a composition comprising a polypeptide and gadopentetate dimeglumine covalentiy attached to the polypeptide. Also provided is a method for protecting gadopentetate dimeglumine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of gadopentetate dimeglumine from a composition comprising covalently attaching it to the polypeptide.

19 CW195P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GADOTERIDOL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to gadoteridol, as well as methods for protecting and administering gadoteridol. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Gadoteridol is a known pharmaceutical agent that is used as a contrast reagent in diagnostic imaging. Its chemical name is (+,-)-[ 10-(2-hydroxypropyl)- 1,4,7, 10- tetraazacyclododecane- 1,4,7- triacetato(3-)] gadolinium. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW195P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW195P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW195P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(gadoteridol) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching gadoteridol to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising gadoteridol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and gadoteridol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Gadoteridol preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW195P

the C-terminus of the poljφeptide. In another preferred embodiment the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting gadoteridol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering gadoteridol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefened embodiment, gadoteridol is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, gadoteridol is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and gadoteridol is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, gadoteridol is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, gadoteridol is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant CW195P

from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching gadoteridol to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, gadoteridol and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW195P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize gadoteridol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of gadoteridol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Gadoteridol is the subject of U.S. Patent Number 4,885,363, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises gadoteridol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW195P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding iii the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are estabUshed during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW195P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected ώ increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW195P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW195P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide canier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW195P

maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, gadoteridol is covalently attached to the polypeptide via a hydroxyl or amino group, or alternatively through an artificial linker.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW195P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-gadoteridol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW195P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW195P

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture re fluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW195P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and

( 5 ^" gadoteridol covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of 10 two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

15 7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein gadoteridol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

20 10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW195P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein gadoteridol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing gadoteridol from said composition in a pH-dependent manner.

19. A method for protecting gadoteridol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of gadoteridol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching gadoteridol to said polypeptide.

21. A method for delivering gadoteridol to a patient comprising administering to said patient a composition comprising: a polypeptide; and gadoteridol covalently attached to said polypeptide.

22. The method of claim 21 wherein gadoteridol is released from said composition by an enzyme-catalyzed release.

17 CW195P

23. The method of claim 21 wherein gadoteridol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW195P

Abstract

A composition comprising a polypeptide and gadoteridol covalently attached to the polypeptide. Also provided is a method for delivery of gadoteridol to a patient comprising administering to the patient a composition comprising a polypeptide and gadoteridol covalently attached to the polypeptide. Also provided is a method for protecting gadoteridol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of gadoteridol from a composition comprising covalently attaching it to the polypeptide.

19 CW196P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GANAXOLONE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to ganaxolone, as well as methods for protecting and administering ganaxolone. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ganaxolone is a known pharmaceutical agent that is used in the treatment of epilepsy and migraine. Its chemical name is (3alpha,5alpha)-3-hydroxy-3-methyl- pregnan-20-one. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor. CW196P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW196P

reproducibility. In addition, encapsulated drags rely on diffusion out ofthe matrix, which is highly dependant on the water solubihty of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW196P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ganaxolone) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ganaxolone to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ganaxolone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ganaxolone covalently attached to the polypeptide. Preferably, the polypeptide is (i)_.an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Ganaxolone preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW196P

carboxylic acid and is covalentiy attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ganaxolone from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ganaxolone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, ganaxolone is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, ganaxolone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and ganaxolone is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, ganaxolone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, ganaxolone is released from the composition in a CW196P

sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ganaxolone to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ganaxolone and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW196P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ganaxolone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ganaxolone. . Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Ganaxolone is the subject of DE 2162555 A (1972), WO 93/3732 (1993) - based on priority US application 745216 (1991), WO 93/5786 1993 - based on priority US application 759512 (1991), and WO 94/27608 (1994) - based on priority US application 68378 (1993), herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises ganaxolone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding CW196P

are defined by the free energy of a particular condition of the protein that rehes on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW196P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW196P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW196P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide canier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW196P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ganaxolone is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW196P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized fransporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of speciaUzed adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ganaxolone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW196P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can_then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW196P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^'

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW196P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ganaxolone covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ganaxolone is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW196P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ganaxolone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ganaxolone from said composition in a pH-dependent manner.

19. A method for protecting ganaxolone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ganaxolone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ganaxolone to said polypeptide.

21. A method for delivering ganaxolone to a patient comprising administering to said patient a composition comprising: a polypeptide; and ganaxolone covalently attached to said polypeptide.

22. The method of claim 21 wherein ganaxolone is released from said composition by an enzyme-catalyzed release.

17 CW196P

23. The method of claim 21 wherein ganaxolone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW196P

Abstract

A composition comprising a polypeptide and ganaxolone covalently attached to the polypeptide. Also provided is a method for delivery of ganaxolone to a patient comprising administering to the patient a composition comprising a polypeptide and ganaxolone covalently attached to the polypeptide. Also provided is a method for protecting ganaxolone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ganaxolone from a composition comprising covalentiy attaching it to the polypeptide.

19 CW197P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GANCICLOVIR AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to ganciclovir, as well as methods for protecting and administering ganciclovir. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ganciclovir is a known pharmaceutical agent that is used in the treatment of cytomegaloviras (CMV) retinitis in immunocompromised patients, including patients with acquired immunodeficiency syndrome (AIDS). It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability

l CW197P

of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stabUity are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified CW197P

amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

. Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that CW197P

incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ganciclovir) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ganciclovir to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechamsm.

Alternatively, the present invention provides a pharmaceutical composition comprising ganciclovir microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ganciclovir covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a CW197P

heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ganciclovir preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ganciclovir from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ganciclovir to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, ganciclovir is released from the composition by CW197P

an enzyme-catalyzed release. In another preferred embodiment, ganciclovir is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and ganciclovir is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, ganciclovir is released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, ganciclovir is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. The method comprises the steps of:

(a) attaching ganciclovir to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ganciclovir and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a CW197P

carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, . incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ganciclovir and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ganciclovir. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Ganciclovir is the subject of U.S. Patent Numbers 4,355,032, 4,423,050, 4,507,305 and 4,642,346, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises ganciclovir covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the CW197P

conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial arrangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only CW197P

5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionizeln the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight CW197P

active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW197P

TABLE

Amino acid MW Active agent MW

Glycine 57, Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terπύnus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

1 1 CW197P

rx)ly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Altematively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW197P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ganciclovir is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW197P

mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ganciclovir conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

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In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW197P

Preparation of γ- Alk l Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of PoIy[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW197P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and ganciclovir covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oUgopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more sjmthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ganciclovir is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW197P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ganciclovir is conformationally ' protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ganciclovir from said composition in a pH-dependent manner.

19. A method for protecting ganciclovir from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of ganciclovir from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching

■ ganciclovir to said polypeptide.

21. A method for delivering ganciclovir to a patient comprising administering to said patient a composition comprising: a poljφeptide; and ganciclovir covalently attached to said polypeptide.

22. The method of claim 21 wherein ganciclovir is released from said composition by an enzyme-catalyzed release.

18 CW197P

23. The method of claim 21 wherein ganciclovir is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and ganciclovir covalently attached to the polypeptide. Also provided is a method for delivery of ganciclovir to a patient comprising administering to the patient a composition comprising a polypeptide and ganciclovir covalently attached to the polypeptide. Also provided is a method for protecting ganciclovir from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ganciclovir from a composition comprising covalently attaching it to the polypeptide.

19 CW198P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GANTOFTBAN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to gantofiban, as well as methods for protecting and administering gantofiban. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Gantofiban is a known pharmaceutical agent that is used in the treatment of thrombosis and angina. Its chemical name is 4-[[(5R)-3-[4-[imino[(methoxycarbonyl) amino]methyl]phenyl]-2-oxo-5-oxazolidinyl]methyl]-l-piperazineacetic acid ethyl ester 2-hydroxy-l,2,3-propanetricarboxylate (1:1. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical CW198P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient comphance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabiUty of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW198P

shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on^* the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW198P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is Umited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (gantofiban) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching gantofiban to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising gantofiban microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and gantofiban covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW199P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting gastrin 17 immunogen from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering gastrin 17 immunogen to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, gastrin 17 immunogen is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, gastrin 17 immunogen is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and gastrin 17 immunogen is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, gastrin 17 immunogen is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefened embodiment, gastrin 17 immunogen is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW199P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching gastrin 17 immunogen to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, gastrin 17 immunogen and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize gastrin 17 immunogen and prevent its digestion in the CW199P

stomach. In addition, the pharmacologic effect can be prolonged by delayed release of gastrin 17 immunogen. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Gastrin 17 immunogen is the subject of U.S. Patent Numbers 5622702, 5785970, 5607676, and 5609870, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises gastrin 17 immunogen covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have

- primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW199P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW199P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in conceit with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW199P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin , (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a poljφeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW199P

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

n CW199P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, gastrin 17 immunogen is covalently attached to the polypeptide via a peptide bond.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW199P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-gastrin 17 immunogen conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product

13 CW199P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyrrolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alk l Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW199P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW199P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and gastrin 17 immunogen covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said poljφeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein gastrin 17 immunogen is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW199P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein gastrin 17 immunogen is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing gastrin 17 immunogen from said composition in a pH-dependent manner.

19. A method for protecting gastrin 17 immunogen from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of gastrin 17 immunogen from a composition wherein said composition comprises a poljφeptide, said method comprising covalently attaching gastrin 17 immunogen to said poljφeptide.

21. A method for delivering gastrin 17 immunogen to a patient comprising administering to said patient a composition comprising: a polypeptide; and gastrin 17 immunogen covalently attached to said polypeptide.

22. The method of claim 21 wherein gastrin 17 immunogen is released from said composition by an enzyme-catalyzed release.

17 CW199P

23. The method of claim 21 wherein gastrin 17 immunogen is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW199P

Abstract

A composition comprising a polypeptide and gastrin 17 immunogen covalently attached to the polypeptide. Also provided is a method for delivery of gastrin 17 immunogen to a patient comprising administering to the patient a composition comprising a polypeptide and gastrin 17 immunogen covalently attached to the polypeptide. Also provided is a method for protecting gastrin 17 immunogen from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of gastrin 17 immunogen from a composition comprising covalently attaching it to the polypeptide.

19 CW200P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GEMCITABINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to gemcitabine, as well as methods for protecting and administering gemcitabine. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness ofthe phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Gemcitabine is a known pharmaceutical agent that is used in the tteatment of — . Its chemical name is . Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW200P

invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the abihty to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW200P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent The peptide-drag conjugates of this class of drug dehvery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW200P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (xxxxx) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching gemcitabine to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising gemcitabine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and gemcitabine covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Gemcitabine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide. CW200P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt^' When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tabbt, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting gemcitabine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering gemcitabine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, gemcitabine is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, gemcitabine is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and gemcitabine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, gemcitabine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, gemcitabine is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW200P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching gemcitabine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, gemcitabine and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize gemcitabine and prevent its digestion in the stomach. In CW200P

addition, the pharmacologic effect can be prolonged by delayed release of gemcitabine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Gemcitabine is the subject of U.S. Patent Number yyyyy, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises gemcitabine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW200P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW200P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino- acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given appUcation. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, lpw molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW200P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin E 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent deUvery composition or over two times the maximum drag loading for dextran. This is only for an NT or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW200P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicUlin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular ttansamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

11 CW200P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, gemcitabine is covalentiy attached to the polypeptide via the zzzzzzz.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW200P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-gemcitabine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product

13 CW200P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW200P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW200P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and gemcitabine covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein gemcitabine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW200P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

(5 14. The composition of claim 1 wherein said composition is in the form of an

₍ ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an 10 oral suspension.

17. The composition of claim 1 wherein gemcitabine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing gemcitabine from said composition in a pH-dependent manner.

15 19. A method for protecting gemcitabine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of gemcitabine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching gemcitabine to said polypeptide.

20 21. A method for delivering gemcitabine to a patient comprising administering to said patient a composition comprising: a polypeptide; and gemcitabine covalently attached to said polypeptide.

22. The method of claim 21 wherein gemcitabine is released from said 25 composition by an enzyme-catalyzed release.

17 CW200P

23. The method of claim 21 wherein gemcitabine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW200P

Abstract

A composition comprising a polypeptide and gemcitabine covalently attached to the polypeptide. Also.provided is a method for delivery of gemcitabine to a patient comprising administering to the patient a composition comprising a polypeptide and gemcitabine covalently attached to the polypeptide. Also provided is a method for protecting gemcitabine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of gemcitabine from a composition comprising covalently attaching it to the polypeptide.

19 CW201P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GEMFIBROZIL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to gemfibrozil, as well as methods for protecting and administering gemfibrozil. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both weU studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Gemfibrozil is a known pharmaceutical agent that is used in the treatment of hyperlipidemia. Its chemical name is 5-(2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted deUvery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW201P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibUity and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW201P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzjmies residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW201P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (gemfibrozil) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching gemfibrozil to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising gemfibrozil microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and gemfibrozil covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Gemfibrozil preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW201P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting gemfibrozil from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering gemfibrozil to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, gemfibrozil is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, gemfibrozil is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and gemfibrozil is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, gemfibrozil is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, gemfibrozil is released from the CW201P

composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching gemfibrozil to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, gemfibrozil and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is CW201P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize gemfibrozil and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of gemfibrozil. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Gemfibrozil is the subject of GB 1225575 (1971), priority based on US application 73046 (1968), herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises gemfibrozil covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW201P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability ofthe protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW201P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophiUcity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW201P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW201P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drug loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide canier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

u CW201P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teiminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, gemfibrozil is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW201P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-gemfibrozil conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW201P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyπolidinopyridine, dimethylaminopyridine. triethylamine or tributylamine.

14 CW201P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodumide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then bestirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- lkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture re fluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^'

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the detaUs within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW201P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and gemfibrozil covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein gemfibrozil is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW201P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein gemfibrozil is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing gemfibrozil from said composition in a pH-dependent manner.

19. A method for protecting gemfibrozil from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of gemfibrozil from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching gemfibrozil to said polypeptide.

21. A method for delivering gemfibrozil to a patient comprising administering to said patient a composition comprising: a polypeptide; and gemfibrozil covalently attached to said polypeptide.

22. The method of claim 21 wherein gemfibrozil is released from said composition by an enzyme-catalyzed release.

17 CW201P

23. The method of claim 21 wherein gemfibrozil is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW201P

Abstract

A composition comprising a polypeptide and gemfibrozil covalently attached to the polypeptide. Also provided is a method for delivery of gemfibrozil to a patient comprising administering to the patient a composition comprising a polypeptide and gemfibrozil covalently attached to the polypeptide. Also provided is a method for protecting gemfibrozil from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of gemfibrozil from a composition comprising covalently attaching it to the polypeptide.

19 CW202P

A NOVEL PHARMACEU ICAL COMPOUND CONTAINING GENTAMICIN ISOTON AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to gentamicin isoton, as well as methods for protecting and administering gentamicin isoton. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Gentamicin isoton is a known pharmaceutical agent that is used in the treatment of bacterial infections and muscular dysttophy. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its structure is:

CW202P

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release CW202P

through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide CW202P

linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (gentamicin isoton) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching gentamicin isoton to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising gentamicin isoton microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and gentamicin isoton covalently attached to the polypeptide. Preferably, the polypeptide is (i) an CW202P

oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Gentamicin isoton preferably is covalentiy attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the poljφeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting gentamicin isoton from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering gentamicin isoton to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently CW202P

attached to the polypeptide. In a prefened embodiment, gentamicin isoton is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, gentamicin isoton is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and gentamicin isoton is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, gentamicin isoton is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, gentamicin isoton is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

I

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching gentamicin isoton to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, gentamicin isoton and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side CW202P

chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize gentamicin isoton and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of gentamicin isoton. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises gentamicin isoton covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture. CW202P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. CW202P

Moreover, protein conformation generally coπTTols the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW202P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW202P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

11 CW202P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, gentamicin isoton is covalently attached to the polypeptide via the hydroxyl group.

The carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW202P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the canier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechamsm of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-gentamicin isoton conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW202P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can-then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or ttibutylamine.

14 CW202P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alk l Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW202P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and gentamicin isoton covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein gentamicin isoton is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW202P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein gentamicin isoton is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing gentamicin isoton from said composition in a pH-dependent manner.

19. A method for protecting gentamicin isoton from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of gentamicin isoton from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching gentamicin isoton to said polypeptide.

21. A method for delivering gentamicin isoton to a patient comprising administering to said patient a composition comprising: a polypeptide; and gentamicin isoton covalently attached to said polypeptide.

22. The method of claim 21 wherein gentamicin isoton is released from said composition by an enzyme-catalyzed release.

17 CW202P

23. The method of claim 21 wherein gentamicin isoton is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW202P

Abstract

A composition comprising a polypeptide and gentamicin isoton covalently attached to the polypeptide. Also provided is a method for deUvery of gentamicin isoton to a patient comprising administering to the patient a composition comprising a polypeptide and gentamicin isoton covalently attached to the polypeptide. Also provided is a method for protecting gentamicin isoton from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for conttolling release of gentamicin isoton from a composition comprising covalently attaching it to the polypeptide.

19 CW203P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GEPIRONE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to gepirone, as well as methods for protecting and administering gepirone. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Gepirone is a known pharmaceutical agent that is used in the treatment of anxiety and depression. Its chemical name is 4,4-dimethyl-l-[4-[4-(2-pyrimidinyl)-l- piperazinyl]butyl]-2,6-piperidinedione. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW203P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase

5 markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf ( life or survival in the stomach, will assure dosage reproducibility and perhaps even

I reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly 10 acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the IS active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and

20 aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can

25 also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW203P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW203P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (gepirone) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalentiy attaching gepirone to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising gepirone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and gepirone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Gepirone preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW203P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting gepirone from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering gepirone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, gepirone is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, gepirone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and gepirone is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, gepirone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, gepirone is released from the composition in a sustained release. CW203P

In yet another prefened embodiment, the composition further comprises an adjuvant covalentiy attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching gepirone to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, gepirone and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is CW203P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize gepirone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of gepirone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Gepirone is the subject of GB 2114122 B (1985), based on priority US application

334688 (1981), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises gepirone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW203P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW203P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or lipophiUcity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but wUl ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW203P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. GeneraUy, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW203P

delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teiminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalentiy attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW203P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release ofthe drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW203P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-gepirone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW203P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW203P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW203P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and gepirone covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein gepirone is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW203P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein gepirone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing gepirone from said composition in a pH-dependent manner.

19. A method for protecting gepirone from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for conttolling release of gepirone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching gepirone to said polypeptide.

21. A method for delivering gepirone to a patient comprising administering to said patient a composition comprising: a polypeptide; and gepirone covalently attached to said polypeptide.

22. The method of claim 21 wherein gepirone is released from said composition by an enzyme-catalyzed release.

17 CW203P

23. The method of claim 21 wherein gepirone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW203P

Abstract

A composition comprising a polypeptide and gepirone covalently attached to the polypeptide. Also provided is a method for delivery of gepirone to a patient comprising administering to the patient a composition comprising a polypeptide and gepirone covalently attached to the polypeptide. Also provided is a method for protecting gepirone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for conttolling release of gepirone from a composition comprising covalently attaching it to the polypeptide.

19 CW204P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GLATIRAMER ACETATE AND METHODS OF MAKING AND USING SAME

FTELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to glatiramer acetate, as well as methods for protecting and administering glatiramer acetate. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Glatiramer acetate is a known pharmaceutical agent that is used in the tteatment of multiple sclerosis. Its chemical name is L-glutamic acid polymer with L-alanine, L- lysine and L-tyrosine, acetate.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique: increasing the stability of the active agent, such as prolonging shelf CW204P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW204P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less, than 5 microns. CW204P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (glatiramer acetate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching glatiramer acetate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising glatiramer acetate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and glatiramer acetate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Glatiramer acetate preferably is covalentiy attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide. CW204P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting glatiramer acetate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering glatiramer acetate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a preferred embodiment, glatiramer acetate is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, glatiramer acetate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and glatiramer acetate is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, glatiramer acetate is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, glatiramer acetate is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW204P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching glatiramer acetate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, glatiramer acetate and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present .invention provides several benefits for active agent delivery. First, the invention can stabilize glatiramer acetate and prevent its digestion in the stomach. In CW204P

addition, the pharmacologic effect can be prolonged by delayed release of glatiramer acetate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Glatiramer acetate is the subject of U.S. Patent Number 6,054,430 and 5,981 ,589, herein incorporated by reference, which describes how to make that drug.

The composition of the invention comprises glatiramer acetate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iu) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial arrangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW204P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of irreversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW204P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

> Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate CW204P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a poljφeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW204P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be. copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

11 CW204P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythrebnine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the G-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, glatiramer acetate is covalently attached to the polypeptide via a peptide bond.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW204P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-glatiramer acetate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

AciόVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with dusopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product

13 CW204P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus ofthe peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or ttibutylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW204P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount Of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW204P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and glatiramer acetate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said poljφeptide is a homopolymer of a namrally occuning amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturaUy occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein glatiramer acetate is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW204P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein glatiramer acetate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing glatiramer acetate from said composition in a pH-dependent manner.

19. A method for protecting glatiramer acetate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of glatiramer acetate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching glatiramer acetate to said polypeptide.

21. A method for delivering glatiramer acetate to a patient comprising administering to said patient a composition comprising: a polypeptide; and glatiramer acetate covalently attached to said polypeptide.

22. The method of claim 21 wherein glatiramer acetate is released from said composition by an enzyme-catalyzed release.

17 CW204P

23. The method of claim 21 wherein glatiramer acetate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW204P

Abstract

A composition comprising a polypeptide and glatiramer acetate covalently attached to the polypeptide. Also provided is a method for delivery of glatiramer acetate to a patient comprising administering to the patient a composition comprising a polypeptide and glatiramer acetate covalently attached to the polypeptide. Also provided is a method for protecting glatiramer acetate from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for confrolling release of glatiramer acetate from a composition comprising covalently attaching it to the polypeptide.

19 CW205P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GLIMEPIRIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to glimepiride, as well as methods for protecting and administering glimepiride. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Glimepiride is a known pharmaceutical agent that is used in the treatment of diabetes. Its chemical name is trans-3-ethyl-2,5-dihydro-4-methyl-N-[2-[4-[[[[(4- methylcyclohexyl)ami no]carbonyl]amino]sulfonyl]phenyl]ethyl]-2-oxo- 1 H-pyrrole- 1 - carboxamide. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW205P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW205P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW205P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (glimepiride) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching glimepiride to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising glimepiride microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and glimepiride covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Glimepiride preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW205P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting glimepiride from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering glimepiride to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, glimepiride is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, glimepiride is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and glimepiride is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, glimepiride is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, glimepiride is released from the composition in a CW205P

sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated info a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching glimepiride to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, glimepiride and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular fransamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is CW205P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize glimepiride and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of glimepiride. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Glimepiride is the subject of U.S. Patent Number 4,379,785, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises glimepiride covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteimine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW205P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are estabUshed during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW205P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW205P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the pnly polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon jnd drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B, (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW205P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydf oxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW205P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, glimepiride is covalentiy attached to the polypeptide via the amine group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW205P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-glimepiride conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW205P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW205P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW205P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and glimepiride covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said poljφeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein glimepiride is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW205P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein glimepiride is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing glimepiride from said composition in a pH-dependent manner.

19. A method for protecting glimepiride from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of glimepiride from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching glimepiride to said polypeptide.

21. A method for delivering glimepiride to a patient comprising administering to said patient a composition comprising: a polypeptide; and glimepiride covalently attached to said polypeptide.

22. The method of claim 21 wherein glimepiride is released from said composition by an enzyme-catalyzed release.

17 CW205P

23. The method of claim 21 wherein glimepiride is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW205P

Abstract

A composition comprising a polypeptide and glimepiride covalently attached to the polypeptide. Also.provided is a method for delivery of glimepiride to a patient comprising administering to the patient a composition comprising a polypeptide and glimepiride covalently attached to the polypeptide. Also provided is a method for protecting glimepiride from degradation comprising covalentiy attaching it to a polypeptide. Also provided is a method for controlling release of glimepiride from a composition comprising covalently attaching it to the polypeptide.

19 CW206P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GLIPIZIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to glipizide, as well as methods for protecting and administering glipizide. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Glipizide is a known pharmaceutical agent that is used in the treatment of diabetes. It is both commercially available and readily manufactured using published sjmthetic schemes by those of ordinary skill in the art. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW206P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW206P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW206P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (glipizide) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching glipizide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising glipizide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and glipizide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Glipizide preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW206P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting glipizide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering glipizide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, glipizide is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, glipizide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and glipizide is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, glipizide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, glipizide is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW206P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching glipizide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, glipizide and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutaπuc acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference. CW206P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize glipizide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of glipizide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises glipizide covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial arrangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW206P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW206P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophiHcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but wUl ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW206P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B^ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

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The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- 5 terminus of the oligopeptide is the preferred point of attachment. If the active agent is an ( amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order

! to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the 10 preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the 15 peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid

20 moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively,

25 the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino

30 acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

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The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, glipizide is covalentiy attached to the polypeptide via the amine group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

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There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal ttansport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-glipizide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product

13 CW206P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW206P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW206P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and glipizide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more sjmthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein glipizide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW206P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein glipizide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing glipizide from said composition in a pH-dependent manner.

19. A method for protecting glipizide from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of glipizide from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching glipizide to said polypeptide.

21. A method for delivering glipizide to a patient comprising administering to said patient a composition comprising: a polypeptide; and glipizide covalently attached to said polypeptide.

22. The method of claim 21 wherein glipizide is released from said composition by an enzyme-catalyzed release.

17 CW206P

23. The method of claim 21 wherein glipizide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW206P

Abstract

A composition comprising a polypeptide and glipizide covalentiy attached to the polypeptide. Also provided is a method for delivery of glipizide to a patient comprising administering to the patient a composition comprising a polypeptide and glipizide covalentiy attached to the polypeptide. Also provided is a method for protecting glipizide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of glipizide from a composition comprising covalentiy attaching it to the polypeptide.

19 CW207P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GLUCAGON AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to glucagon, as well as methods for protecting and administering glucagon. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Glucagon is a known pharmaceutical agent that is used in the treatment of diabetes It is a namrally occurring peptide that can either be isolated or synthesized, preferably using recombinant DNA technology.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW207P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW207P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use -has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral admimstration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW207P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (glucagon) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching glucagon to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. . In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzjmiatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising glucagon microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and glucagon covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Glucagon preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another preferred embodiment the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide. CW207P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tab'εt, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting glucagon from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering glucagon to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a preferred embodiment, glucagon is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, glucagon is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and glucagon is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, glucagon is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, glucagon is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW207P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching glucagon to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, glucagon and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize glucagon and prevent its digestion in the stomach. In addition, CW207P

the pharmacologic effect can be prolonged by delayed release of glucagon. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises glucagon covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial arrangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW207P

and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are 5 "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and ζ contribute to the overall stabiUty of the folded protein where the degree to which ideal _f packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

10 Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabUization of a protein. Typically, the folded state of a protein is favored by only

15 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

20 Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For 25 instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will CW207P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can aU be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechamsm. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the CW207P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order

10 CW207P

to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechamsm can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

1 1 CW207P

preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyI-3-acyltriazenes to tefragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, glucagon is covalentiy attached to the polypeptide via a peptide linkage.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

12 CW207P

intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-glucagon conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

13 CW207P

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutaπuc acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

14 CW207P

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AIkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overmght). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW207P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and

( 5 glucagon covalentiy attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of 10 two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

15 7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

_*

8. The composition of claim 1 wherein glucagon is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a πucroencapsulating agent.

20 10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW207P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein glucagon is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing glucagon from said composition in a pH-dependent manner.

19. A method for protecting glucagon from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of glucagon from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching glucagon to said polypeptide.

21. A method for delivering glucagon to a patient comprising administering to said patient a composition comprising: a polypeptide; and glucagon covalently attached to said polypeptide.

22. The method of claim 21 wherein glucagon is released from said composition by an enzyme-catalyzed release.

17 CW207P

23. The method of claim 21 wherein glucagon is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW207P

Abstract

A composition comprising a polypeptide and glucagon covalently attached to the polypeptide. Also proyided is a method for delivery of glucagon to a patient comprising administering to the patient a composition comprising a polypeptide and glucagon covalently attached to the polypeptide. Also provided is a method for protecting glucagon from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of glucagon from a composition comprising covalentiy attaching it to the polypeptide.

19 CW208P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GLYBURIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to glyburide, as well as methods for protecting and administering glyburide. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Glyburide is a sulfonylurea antidiabetic agent used in the treatment of diabetes. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW208P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabUity of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW208P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW208P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (glyburide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching glyburide to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal ttact, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action inttoduces a second order sustained release mechamsm.

Alternatively, the present invention provides a pharmaceutical composition comprising glyburide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and glyburide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropoljmier of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Glyburide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the poljφeptide. In a prefened embodiment, the active agent is a CW208P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another 5 preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

(

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino 10 acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationaUy protected by folding of the polypeptide about the active agent. In 15 another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting glyburide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering glyburide to a patient, the 20 patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, glyburide is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, glyburide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. 25 In another prefened embodiment, the composition further comprises a microencapsulating agent and glyburide is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, glyburide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, glyburide is released from the composition in a sustained release. CW208P

In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching glyburide to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, glyburide and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis ofthe polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transaπunation. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW208P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize glyburide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of glyburide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises glyburide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW208P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW208P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected tδ increase the hydropnilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW208P

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin. Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW208P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

U CW208P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechamsm can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking orgamc compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, glyburide is covalently attached to the polypeptide via the amine group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW208P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal 5 epithelia efficiently via specialized transporters. The entire membrane ttansport system is r intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect

I that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the

10 transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active

15 agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the 20 catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-glyburide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

25 Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW208P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW208P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with dHsopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture re fluxed under a nifrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^•

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW208P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and glyburide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more sjmthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein glyburide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW208P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein glyburide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing glyburide from said composition in a pH-dependent manner.

19. A method for protecting glyburide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of glyburide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching glyburide to said polypeptide.

21. A method for delivering glyburide to a patient comprising administering to said patient a composition comprising: a polypeptide; and glyburide covalently attached to said polypeptide.

22. The method of claim 21 wherein glyburide is released from said composition by an enzyme-catalyzed release.

17 CW208P

23. The method of claim 21 wherein glyburide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW208P

Abstract

A composition comprising a polypeptide and glyburide covalently attached to the polypeptide. Also provided is a method for delivery of glyburide to a patient comprising administering to the patient a composition comprising a polypeptide and glyburide covalently attached to the polypeptide. Also provided is a method for protecting glyburide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for conttolling release of glyburide from a composition comprising covalently attaching it to the polypeptide.

19 CW209P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING GRANISETRON AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to granisetron, as well as methods for protecting and administering granisetron. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Granisetron is a known phaimaceutical agent that is used in the treatment of nausea and vomiting in cancer patients Its chemical name is endo-l-methyl-N-(9- methyl-9-azabicyclo[3,3,l]non-3-yl)-lH-indazole-3-carboxamide. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW209P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI ttact, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW209P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nifrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to freat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW209P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (granisetron) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching granisetron to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechamsm.

Alternatively, the present invention provides a phaimaceutical composition comprising granisetron microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and granisetron covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Granisetron preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW209P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the poljφeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting granisetron from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering granisetron to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a poljφeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, granisetron is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, granisetron is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and granisetron is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, granisetron is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, granisetron is released from the composition in a CW209P

sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising _, polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching granisetron to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, granisetron and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW209P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize granisetron and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of granisetron. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal ttact can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Granisetron is the subject of U.S. Patent Number 4,886,808, herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises granisetron covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW209P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining" forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW209P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW209P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) ' 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW209P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminiis examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

n CW209P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system. ^'

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Altematively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, granisetron is covalently attached to the polypeptide via the amine group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW209P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the ttansported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-granisetron conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW209P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can- then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW209P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. - The solution can then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^■

Preparation of Poly[γ-Alkyl Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW209P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and granisetron covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein granisetron is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW209P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 fiirther comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein granisetron is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing granisetron from said composition in a pH-dependent manner.

19. A method for protecting granisetron from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of granisetron from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching granisetron to said polypeptide.

21. A method for delivering granisetton to a patient comprising administering to said patient a composition comprising: a polypeptide; and granisetron covalently attached to said polypeptide.

22. The method of claim 21 wherein granisetron is released from said composition by an enzyme-catalyzed release.

17 CW209P

23. The method of claim 21 wherein granisetton is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW209P

Abstract

A composition comprising a polypeptide and granisetton covalently attached to the polypeptide. Also provided is a method for delivery of granisetron to a patient comprising administering to the patient a composition comprising a polypeptide and granisetron covalently attached to the polypeptide. Also provided is a method for protecting granisetton from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of granisetron from a composition comprising covalently attaching it to the polypeptide.

19 CW210P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING HALOPERIDAL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to haloperidal, as well as methods for protecting and administering haloperidal. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Haloperidal is a known phaimaceutical agent that is used in the treatment of psychotic disorders. It is both commercially available and readily manufactured using published synthetic schemes by those of ordinary skill in the art. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW210P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly- acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW210P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW210P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(haloperidal) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching haloperidal to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising haloperidal microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and haloperidal covalentiy attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Haloperidal preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW210P

the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalentiy attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting haloperidal from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering haloperidal to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefeoed embodiment, haloperidal is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, haloperidal is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and haloperidal is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, haloperidal is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, haloperidal is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant CW210P

from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching haloperidal to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA):

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, haloperidal and a second active agent can be copolymerized in step (c). In another preferred embodiment the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, argimne, asparagine, cysteine, lysine, threomne, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW210P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize haloperidal and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of haloperidal. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention ako allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises haloperidal covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ti) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stracmres. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and rums. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial anangement of the side chains constimte the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW210P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW210P

enriched in the amino acids in the table provided below. Polar amino acids, oh the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW210P

weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW210P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

u CW210P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary ttact can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking orgamc compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, haloperidal is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW210P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized ttansporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-haloperidal conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0 C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product

13 CW210P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW210P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/CTerminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole ⁽ followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for ' several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be Hmited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW210P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and haloperidal covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more sjmthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is. a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein haloperidal is covalently attached to a side chain, the N-terminus or the C-terminus of said poljφeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant

16 CW210P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein haloperidal is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing haloperidal from said composition in a pH-dependent manner.

19. A method for protecting haloperidal from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of haloperidal from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching haloperidal to said polypeptide.

21. A method for delivering haloperidal to a patient comprising administering to said patient a composition comprising: a polypeptide; and haloperidal covalently attached to said polypeptide.

22. The method of claim 21 wherein haloperidal is released from said composition by an enzyme-catalyzed release.

17 CW210P

23. The method of claim 21 wherein haloperidal is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW210P

Abstract

A composition comprising a polypeptide and haloperidal covalently attached to the polypeptide. Also provided is a method for delivery of haloperidal to a patient comprising administering to the patient a composition comprising a polypeptide and haloperidal covalently attached to the polypeptide. Also provided is a method for protecting haloperidal from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of haloperidal from a composition comprising covalently attaching it to the polypeptide.

19 CW211P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING AND METHODS

OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to a quinolone antibiotic, as well as methods for protecting and administering a quinolone antibiotic. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

The quinolone antibiotic of the present invention is a known phaimaceutical agent that is used in the treatment of bacterial infections. Its chemical name is l-cyclopropyl-8- (difluoromethoxy)-7-[( 1 R)-2,3-dihydro- 1 -methyl- 1 H-isoindol-5-yl]- 1 ,4-dihydro-4-oxo-3- quinolinecarboxylic acid monomethanesulfonate. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor. CW211P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW211P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreUable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent deUvery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW211P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. . Particle size not only becomes a problem with injectable drags, as in the H AR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (a quinolone antibiotic) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching a quinolone antibiotic to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechamsm.

Alternatively, the present invention provides a phaimaceutical composition comprising a quinolone antibiotic microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and a quinolone antibiotic covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

A quinolone antibiotic preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active CW211P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter. ?

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting a quinolone antibiotic from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering a quinolone antibiotic to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a poljφeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, a quinolone antibiotic is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, a quinolone antibiotic is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and a quinolone antibiotic is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, a quinolone antibiotic is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, a quinolone CW211P

antibiotic is released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching a quinolone antibiotic to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, a quinolone antibiotic and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inttamolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is CW211P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize a quinolone antibiotic and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of a quinolone antibiotic. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

A quinolone antibiotic is the subject of EP 882725 Al (1998), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises a quinolone antibiotic covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW211P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining' forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a . peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW211P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neuttal charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW211P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW211P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW211P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release ofthe drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, a quinolone antibiotic is covalently attached to the polypeptide via the amine group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW211P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-a quinolone antibiotic conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW211P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can. then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- - hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triefhylamine or tributylamine.

14 CW211P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be freated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW211P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and a quinolone antibiotic covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occuning amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein a quinolone antibiotic is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW211P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein a quinolone antibiotic is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing a quinolone antibiotic from said composition in a pH-dependent manner.

19. A method for protecting a quinolone antibiotic from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for conttolling release of a quinolone antibiotic from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching a quinolone antibiotic to said polypeptide.

21. A method for delivering a quinolone antibiotic to a patient comprising administering to said patient a composition comprising: a polypeptide; and a quinolone antibiotic covalently attached to said polypeptide.

22. The method of claim 21 wherein a quinolone antibiotic is released from said composition by an enzyme-catalyzed release.

17 CW211P

23. The method of claim 21 wherein a quinolone antibiotic is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW211P

Abstract

A composition comprising a polypeptide and a quinolone antibiotic covalently attached to the polypeptide. Also provided is a method for delivery of a quinolone antibiotic to a patient comprising administering to the patient a composition comprising a polypeptide and a quinolone antibiotic covalently attached to the polypeptide. Also provided is a method for protecting a quinolone antibiotic from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of a quinolone antibiotic from a composition comprising covalently attaching it to the polypeptide.

19 CW212P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING

HYDROCHLOROTHIAZIDE AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to hydrochlorothiazide, as well as methods for protecting and administering hydrochlorothiazide. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Hydrochlorothiazide is a known pharmaceutical agent that is used in the treatment of hypertension. The chemical stracmre of hydrochlorothiazide is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW212P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase 5 markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf

( life or survival in the stomach, will assure dosage reproducibility and perhaps even

( reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly 10 acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the 15 active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and

20 aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can

25 also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW212P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspaitic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag deUvery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW212P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absorption through the brash-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (hydrochlorothiazide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalentiy attaching hydrochlorothiazide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising hydrochlorothiazide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and hydrochlorothiazide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Hydrochlorothiazide preferably is covalently attached to a side chain, the N- terπύnus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active CW212P

agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting hydrochlorothiazide from degradation comprising covalently attaching it to a polypeptide. •

The invention also provides a method for delivering hydrochlorothiazide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, hydrochlorothiazide is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, hydrochlorothiazide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and hydrochlorothiazide is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment hydrochlorothiazide is released from the composition by a pH- dependent unfolding of the polypeptide. In another preferred embodiment, CW212P

hydrochlorothiazide is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising i polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching hydrochlorothiazide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, hydrochlorothiazide and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW212P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize hydrochlorothiazide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of hydrochlorothiazide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises hydrochlorothiazide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more sjmthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oHgopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW212P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inframolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein, he result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW212P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW212P

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexfran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexfran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW212P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, arnine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric umt forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-termiήus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW212P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechamsm of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these . polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking orgamc compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, hydrochlorothiazide is covalently attached to the polypeptide via the amine group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW212P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-hydrochlorothiazide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

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Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can-then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylaπύne.

14 CW212P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^•

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW212P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and hydrochlorothiazide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein hydrochlorothiazide is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW212P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein hydrochlorothiazide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing hydrochlorothiazide from said composition in a pH-dependent manner.

19. A method for protecting hydrochlorothiazide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of hydrochlorothiazide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching hydrochlorothiazide to said polypeptide.

21. A method for delivering hydrochlorothiazide to a patient comprising administering to said patient a composition comprising: a polypeptide; and hydrochlorothiazide covalently attached to said polypeptide.

22. The method of claim 21 wherein hydrochlorothiazide is released from said composition by an enzyme-catalyzed release.

17 CW212P

23. The method of claim 21 wherein hydrochlorothiazide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW212P

Abstract

A composition comprising a polypeptide and hydrochlorothiazide covalently attached to the polypeptide. Also provided is a method for delivery of hydrochlorothiazide to a patient comprising administering to the patient a composition comprising a polypeptide and hydrochlorothiazide covalently attached to the polypeptide. Also provided is a method for protecting hydrochlorothiazide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release Of hydrochlorothiazide from a composition comprising covalently attaching it to the polypeptide.

19 CW213P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING HYDROCHLOROTHIAZIDE AND TRIAMTERENE AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to hydrochlorothiazide and triamterene, as well as methods for protecting and administering hydrochlorothiazide and triamterene. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Hydrochlorothiazide and triamterene are known phaimaceutical agents that are used together in the treatment of edema and hypertension. The chemical stracmre of triamterene is:

Its chemical name is . The chemical stracture of hydrochlorothiazide is:

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The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human CW213P

growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incorporation of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nittogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the CW213P

active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drugs, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(hydrochlorothiazide and triamterene) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching hydrochlorothiazide and triamterene to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising hydrochlorothiazide and triamterene microencapsulated by a polypeptide. CW213P

The invention provides a composition comprising a polypeptide and hydrochlorothiazide and triamterene covalently attached to the poljφeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Hydrochlorothiazide and triamterene preferably is covalentiy attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalentiy attached to the C-terminus of the polypeptide. In another preferred embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting hydrochlorothiazide and triamterene from degradation comprising covalently attaching it to a polypeptide. CW213P

The invention also provides a method for delivering hydrochlorothiazide and triamterene to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a preferred embodiment, hydrochlorothiazide and triamterene are released from the composition by an enzyme-catalyzed release. In another prefened embodiment, hydrochlorothiazide and triamterene are released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and hydrochlorothiazide and triamterene are released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, hydrochlorothiazide and triamterene are released from the composition by a pH-dependent unfolding of the polypeptide. In another preferred embodiment, hydrochlorothiazide and triamterene are released from the composition in a sustained release. In yet another preferred embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching hydrochlorothiazide and triamterene to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, hydrochlorothiazide and triamterene and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis CW213P

of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize hydrochlorothiazide and triamterene and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of hydrochlorothiazide and triamterene. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises hydrochlorothiazide and triamterene covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW213P

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enttopy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and inttamolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent. CW213P

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophiHcity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide. CW213P

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dexttan, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

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TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

1 1 CW213P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The.product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can ttanslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW213P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, hydrochlorothiazide and triamterene are covalently attached to the polypeptide via the amine group on each.

The polypeptide canier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW213P

mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-hydrochlorothiazide and triamterene conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stined for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0 C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

14 CW213P

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroforπrate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nittogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tefrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alky I Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW213P

Preparation of γ- Alkyl Glutamate-NCA.

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AlkyI Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW213P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and hydrochlorothiazide and triamterene covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein hydrochlorothiazide and triamterene are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW213P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein hydrochlorothiazide and triamterene are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing hydrochlorothiazide and triamterene from said composition in a pH-dependent manner.

19. A method for protecting hydrochlorothiazide and triamterene from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of hydrochlorothiazide and triamterene from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching hydrochlorothiazide and triamterene to said polypeptide.

21. A method for delivering hydrochlorothiazide and triamterene to a patient comprising administering to said patient a composition comprising: a polypeptide; and hydrochlorothiazide and triamterene covalently attached to said polypeptide.

22. The method of claim 21 wherein hydrochlorothiazide and triamterene are released from said composition by an enzyme-catalyzed release.

18 CW213P

23. The method of claim 21 wherein hydrochlorothiazide and triamterene are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and hydrochlorothiazide and triamterene covalently attached to the polypeptide. Also provided is a method for delivery of hydrochlorothiazide and triamterene to a patient comprising administering to the patient a composition comprising a polypeptide and hydrochlorothiazide and triamterene covalently attached to the polypeptide. Also provided is a method for protecting hydrochlorothiazide and triamterene from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of hydrochlorothiazide and triamterene from a composition comprising covalently attaching > it to the polypeptide.

19 CW214P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING HYDROMORPHONE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to hydromoφhone, as well as methods for protecting and administering hydromoφhone. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combimng it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Hydromoφhone is a known pharmaceutical agent that is used in the treatment of cough and pain. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW214P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeabiUty of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW214P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalentiy attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW214P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (hydromoφhone) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching hydromoφhone to the N-terminus, the C-terminus or directiy to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechamsm.

Alternatively, the present invention provides a pharmaceutical composition comprising hydromoφhone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and hydromoφhone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a sjmthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Hydromoφhone preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW214P

carboxylic acid and is covalently attached to the N-terminus ofthe polypeptide. In another preferred embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting hydromoφhone from degradation comprising covalentiy attaching it to a polypeptide.

The invention also provides a method for delivering hydromoφhone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to Λe polypeptide. In a prefened embodiment, hydromoφhone is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, hydromoφhone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and hydromoφhone is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, hydromoφhone is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, hydromoφhone is CW214P

released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching hydromoφhone to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, hydromoφhone and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW214P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize hydromoφhone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of hydromoφhone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises hydromoφhone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturaUy occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW214P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational enfropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW214P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine cany a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent. CW214P

Dexfran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin _t, (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW214P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide canier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inframolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW214P

maximum drag loading ofthe carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, hydromoφhone is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW214P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal , epithelia efficiently via specialized ttansporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system wiU involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal fransport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-hydromoφhone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW214P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nittogen and cooled to 0°C. The solution can- then be tteated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW214P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitatedOut in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW214P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and hydromoφhone covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a sjmthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more sjmthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein hydromoφhone is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW214P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein hydromoφhone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing hydromoφhone from said composition in a pH-dependent manner.

19. A method for protecting hydromoφhone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of hydromoφhone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching hydromoφhone to said polypeptide.

21. A method for delivering hydromoφhone to a patient comprising administering to said patient a composition comprising: a polypeptide; and hydromoφhone covalently attached to said polypeptide.

22. The method of claim 21 wherein hydromoφhone is released from said composition by an enzyme-catalyzed release.

17 CW214P

23. The method of claim 21 wherein hydromoφhone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW214P

Abstract

A composition comprising a polypeptide and hydromoφhone covalently attached to the polypeptide. Also provided is a method for delivery of hydromoφhone to a patient comprising administering to the patient a composition comprising a polypeptide and hydromoφhone covalently attached to the polypeptide. Also provided is a method for protecting hydromoφhone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of hydromoφhone from a composition comprising covalentiy attaching it to the polypeptide.

19 CW215P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING

HYDROXYCHLOROQUINONE AND METHODS OF

MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalentiy attached to hydroxychloroquinone, as well as methods for protecting and administering hydroxychloroquinone. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both weU smdied and occupies a known segment of the phaonaceutical market, and combimng it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Hydroxychloroquinone is a known pharmaceutical agent that is used in the treatment of malaria. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pha naceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken CW215P

under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabUity of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent CW215P

in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incorporates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR CW215P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE ENVENTION

The present invention provides covalent attachment of the active agent (hydroxychloroquinone) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching . hydroxychloroquinone to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, deUvery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechamsm.

Alternatively, the present invention provides a pharmaceutical composition comprising hydroxychloroquinone microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and hydroxychloroquinone covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Hydroxychloroquinone preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent CW215P

is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting hydroxychloroquinone from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering hydroxychloroquinone to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a preferred embodiment, hydroxychloroquinone is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, hydroxychloroquinone is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and hydroxychloroquinone is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, hydroxychloroquinone is released from the composition by a pH- dependent unfolding of the polypeptide. In another preferred embodiment, hydroxychloroquinone is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled CW215P

by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention -also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching hydroxychloroquinone to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, hydroxychloroquinone and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident inframolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW215P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize hydroxychloroquinone and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of hydroxychloroquinone. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises hydroxychloroquinone covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW215P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational . entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW215P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neufral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threomne and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dexttan conjugate CW215P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dexfran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brash-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B$ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW215P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalentiy attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an inttamolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Altematively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW215P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalentiy attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking orgamc compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, hydroxychloroquinone is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW215P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane ttansport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substtate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly preferred when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-hydroxychloroquinone conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW215P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nifrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or ttibutylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW215P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C^'Terminus Conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW215P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and hydroxychloroquinone covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said poljφeptide is a heteropoljmier of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein hydroxychloroquinone is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW215P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein hydroxychloroquinone is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing hydroxychloroquinone from said composition in a pH-dependent manner.

19. A method for protecting hydroxychloroquinone from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controUing release of hydroxychloroquinone from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching hydroxychloroquinone to said polypeptide.

21. A method for delivering hydroxychloroquinone to a patient comprising administering to said patient a composition comprising: a polypeptide; and hydroxychloroquinone covalentiy attached to said polypeptide.

22. The method of claim 21 wherein hydroxychloroquinone is released from said composition by an enzyme-catalyzed release.

17 CW215P

23. The method of claim 21 wherein hydroxychloroquinone is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW215P

Abstract

A composition comprising a polypeptide and hydroxychloroquinone covalently attached to the polypeptide. Also provided is a method for delivery of hydroxychloroquinone to a patient comprising admimstering to the patient a composition comprising a polypeptide and hydroxychloroquinone covalently attached to the polypeptide. Also provided is a method for protecting hydroxychloroquinone from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of hydroxychloroquinone from a composition comprising covalently attaching it to the polypeptide.

19 CW216P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING IBUPROFEN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ibuprofen, as weU as methods for protecting and administering ibuprofen. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ibuprofen is a known pharmaceutical agent that is used in the treatment of pain and arthritis. Its stracture is:

CH₃CHCH₂

--o( ) >--CHCOOH

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken CW216P

under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals purportedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent CW216P

in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directiy to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into me bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR CW216P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE-fNVENTION

The present invention provides covalent attachment of the active agent (ibuprofen) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ibuprofen to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ibuprofen microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ibuprofen covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ibuprofen preferably is covalently attached to a side chain, Λe N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is CW216P

an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a πucroencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ibuprofen from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ibuprofen to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalentiy attached to the polypeptide. In a prefened embodiment, ibuprofen is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, ibuprofen is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and ibuprofen is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, ibuprofen is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, ibuprofen is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW216P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ibuprofen to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ibuprofen and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, argimne, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another preferred embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference. CW216P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ibuprofen and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ibuprofen. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ibuprofen covalentiy attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracmre and the spatial anangement of the side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW216P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, conflgurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stabiUty of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW216P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino. acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine cany a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and argimne, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be Optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW216P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly Umited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B$ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of me active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW216P

The alcohol, amine or carboxylic acid group of an active agent may be covalentiy attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the preferred point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the preferred point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to me C-terminus of e peptide carrier. Because e glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and Λe drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW216P

The invention also provides a method of imparting the same mechamsm of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can franslate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, e active agent can be attached directiy to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to Λe N-terminus, e C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin. As anomer example, mere are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ibuprofen is covalently attached to Λe polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A preferred technique is copolymerization of mixmres of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyeΛylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of Ae invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW216P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane ttansport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the fransported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of fransport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial fransport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of me active agent. Addition of an adjuvant is particularly preferred when using an omerwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into me lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ibuprofen conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW216P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can Λen be stined for several hours at room temperamre, the urea by-product filtered off, and e product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with e N-terminus of me peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stirred at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW216P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C'Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by e γ-alkyl glutamate bioactive agent. The reaction can then be stirred for several hours at room temperamre, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nittogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate e NCA product, which is filtered, dried and recrystallized from a suitable solvent

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW216P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ibuprofen covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more namrally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synmetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synmetic amino acids.

8. The composition of claim 1 wherein ibuprofen is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10: The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW216P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in e form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in me form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in e form of an oral suspension.

17. The composition of claim 1 wherein ibuprofen is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ibuprofen from said composition in a pH-dependent manner.

19. A method for protecting ibuprofen from degradation comprising covalentiy attaching said active agent to a polypeptide.

20. A method for controlling release of ibuprofen from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ibuprofen to said polypeptide.

21. A method for delivering ibuprofen to a patient comprising administering to said patient a composition comprising: a polypeptide; and ibuprofen covalently attached to said polypeptide.

22. The method of claim 21 wherein ibuprofen is released from said composition by an enzyme-catalyzed release.

17 CW216P

23. The method of claim 21 wherein ibuprofen is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW216P

Abstract

A composition comprising a polypeptide and ibuprofen covalently attached to the poljφeptide. Also provided is a method for delivery of ibuprofen to a patient comprising administering to the patient a composition comprising a polypeptide and ibuprofen covalently attached to the polypeptide. Also provided is a method for protecting ibuprofen from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for conttolling release of ibuprofen from a composition comprising covalently attaching it to the polypeptide.

19 CW217P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING IDARUBICIN AND METHODS OF MAKING AND USING SAME

FTELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to idarubicin, as well as methods for protecting and administering idarubicin. This novel compound, referred to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound mat enhances the usefulness of the phaimaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Idarubicin is a known pharmaceutical agent mat is used in me treatment of cancer. Its chemical name is (7S,9S)-9-acetyl-7-[(3-amino-2,3,6-trideoxy-alfa-L-lyxo- hexopyranosyl)oxy]-7,8,9,10-tettahydro-6,9,l l-trihydroxy-5,12-naphthacenedioone. Its structure is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW217P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of mese systems becomes magnified when patient compliance and active agent stability are taken under consideration, for instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibUity and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by e harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeabiUty of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of e active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in e stomach.

Active agent delivery systems also provide e ability to control Λe release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW217P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release Λe active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require me use of spacer groups between me amino acid pendant group and e active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to Λe gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to Λe gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid wiΛout a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in Λe large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where Λe active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW217P

Λe colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem wiΛ injectable drags, as in Λe HAR application, but absoφtion through the brush-border membrane of Λe intestines is limited to less Λan 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (idarubicin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching idarubicin to Λe N- terminus, Λe C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a canier peptide. In certain applications, Λe polypeptide will stabilize the active agent, primarily in Λe stomach, through conformational protection. In these applications, delivery of e active agent is controlled, in part, by the kinetics of unfolding of e carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by Λe body by selectively hydrolyzing Λe peptide bonds of the carrier peptide. This enzymatic action infroduces a second order sustained release mechanism.

Alternatively, Λe present invention provides a pharmaceutical composition comprising idarubicin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and idarubicin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synΛetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synΛetic amino acids.

Idarubicin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW217P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In anoΛer prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, Λe active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another preferred embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyeΛylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in Λe composition, the adjuvant preferably activates an intestinal transporter.

Preferably, Λe composition of the invention is in Λe form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing Λe active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting idarubicin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering idarubicin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, idarubicin is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, idarubicin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and idarubicin is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, idarubicin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, idarubicin is released from the composition in a sustained release. CW217P

In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by me polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalentiy attached to Λe polypeptide. The method comprises Λe steps of:

(a) attaching idarubicin to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing Λe active agent/amino acid complex N-carboxyanhydride (NCA).

In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) wiΛ a second active agent. When steps (a) and (b) are repeated prior to step (c) wiΛ a second agent, idarubicin and a second active agent can be copolymerized in step (c). In another preferred embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of Λe polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular ttansamination. In anoΛer prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulΛydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW217P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incorporated herein by reference.

DETATI F.D DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize idarubicin and prevent its digestion in Λe stomach. In addition, Λe pharmacologic effect can be prolonged by delayed release of idarubicin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of Λe active agent in Λe intestinal fract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Idarubicin is Λe subject of GB 1467383 (1977), herein incorporated by reference, which describes how to make that drag.

The composition of the invention comprises idarubicin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of Λe twenty namrally occurring amino acids, (iii) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synΛetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synΛetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids Λat have primary, secondary and tertiary stractures. The secondary stracture of the protein is Λe local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial anangement of Λe side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW217P

acid residues packing into a hydrophobic core. The amino acid side chains inside Λe protein core occupy Λe same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so Λe best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to Λe thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and Λe hydrophobic effect. Considering protein stability, Λe hydrophobic effect refers to Λe energetic consequences of removing apolar groups from Λe protein interior and exposing them to water. Comparing Λe energy of amino acid hydrolysis wiΛ protein unfolding in the solid reference state, the hydrophobic effect is Λe dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at Λe expense of hydrogen bonds wiΛ water. Water molecules are "pushed out" of Λe packed, hydrophobic protein core. All of Λese forces combine and contribute to Λe overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of Λe protein. The result of maximum packing is to produce a center of residues or hydrophobic core Λat has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drug can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating Λe amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW217P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, Λen Λe carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on Λe other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neufral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of Λe peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of Λe linear sequence can influence how Λese interactions can be maximized and is important in directing the secondary and tertiary stracmres of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incorporated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with Λe kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that Λe kinetics of CW217P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between Λe carrier peptide and the active agent.

Dexttan is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of Λe total drag-dextran conjugate weight wiΛ drag. As stated earlier, polysaccharides are digested mainly in Λe colon and drag absoφtion is mainly limited to Λe colon. As compared to dextran, Λis invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated wiΛ Λe brush-border membrane and so active agent release and subsequent absoφtion can occur in Λe jejunum or Λe ileum. Second, the molecular weight of the carrier molecule can be confroUed and, thus, active agent loading can also be controlled.

As a practical example, the following table lists Λe molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin Be (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through Λe stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW217P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, Λe C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on Λe functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then Λe N- terminus of the oligopeptide is the preferred point of attachment. If Λe active agent is an amine (e.g., ampicillin), then the C-terminus is Λe preferred point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-teπninus examples, Λe peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If Λe active agent is an alcohol, then either the C-terminus or the N-terminus is Λe preferred point of attachment in order to achieve a stable composition. As in e example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to Λe reaction of Λis key intermediate wiΛ the N-terminus of the peptide carrier. The active ingredient can be released from Λe peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to Λe C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

n CW217P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized wiΛ the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by Λe glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from Λe prior art by virtue of the fact that Λe primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some oΛer indigenous enzymes in Λe alimentary tract can affect release.

The active agent can be covalently attached to Λe N-terminus, Λe C-terminus or the side chain of Λe polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As anoΛer example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, idarubicin is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW217P

The addition of stabilizers to the composition has me potential of stabilizing me polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In anoΛer embodiment of Λe invention, a pre-first order release of the active agent is imparted by microencapsulating Λe carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane fransport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect Λat excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to Λe physical properties of Λe ttansported subsfrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein fransport systems and each has its own associated mechanism of ttansport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting Λe mechanisms for intestinal epithelial ttansport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance Λe bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of Λe peptides.

Preferably, the resultant peptide-idarabicin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW217P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nifrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can Λen be stirred for several hours at room temperamre, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stirred for several hours at room temperature, e urea by-product filtered off, and e product precipitated out in eΛer and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In Λe following example the combination of the alcohol wiΛ triphosgene produces a chloroformate, which when reacted with the N-terminus of Λe peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated wiΛ triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is Λen added slowly and the solution stined at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pynolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW217P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can Λen be stirred for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in eΛer and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture re fluxed under a nitrogen atmosphere until me mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW217P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and idarabicin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a namrally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synmetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synmetic amino acids.

8. The composition of claim 1 wherein idarabicin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW217P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in me form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in Λe form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein idarabicin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing idarabicin from said composition in a pH-dependent manner.

19. A method for protecting idarabicin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of idarabicin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching idarabicin to said polypeptide.

21. A method for delivering idarabicin to a patient comprising admimstering to said patient a composition comprising: a polypeptide; and idarabicin covalently attached to said polypeptide.

22. The method of claim 21 wherein idarabicin is released from said composition by an enzyme-catalyzed release.

17 CW217P

23. The method of claim 21 wherein idarabicin_»is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW217P

Abstract

A composition comprising a polypeptide and idarabicin covalently attached to Λe polypeptide. Also provided is a method for delivery of idarabicin to a patient comprising administering to the patient a composition comprising a polypeptide and idarabicin covalentiy attached to the polypeptide. Also provided is a method for protecting idarabicin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of idarabicin from a composition comprising covalently attaching it to Λe polypeptide.

19 CW218P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ILODECAKTN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound Λat comprises a polypeptide Λat is preferably covalentiy attached to ilodecakin, as well as methods for protecting and administering ilodecakin. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well smdied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound at enhances the usefulness of me pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Uodecakin is a known pharmaceutical agent at is used in Λe treatment of hepatitis, autoimmune disorders and HIV infections. Its chemical name is interleukin 10, and it is bom isolatable from natural sources and capable of being synthesized by those of skill in me art.

The novel pharmaceutical compound of me present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of Λese systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another CW218P

invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI fract, permeability of cellular membranes and transport across lipid bilayers. Incorporating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of Λe active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in me stomach.

Active agent delivery systems also provide the ability to control the release of Λe active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed wiΛ a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW218P

unreliable. For example, an enterically coated active agent depends on pH to release Λe active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require Λe use of spacer groups between Λe amino acid pendant group and Λe active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for Λe release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to Λe gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drug deUvery system where the drag is released by bacterial hydrolytic enzymes residing in Λe large intestines. The released dexamethasone active agent, in mm, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines me advantages of covalent drag attachment wiΛ liposome formation where me active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, Λere has been no drag delivery system, heretofore reported, that incorporates e concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW218P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ilodecakin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ilodecakin to the N- terminus, Λe C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, Λe polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In Λese applications, delivery of the active agent is controlled, in part, by Λe kinetics of unfolding of the carrier peptide. Upon entry into Λe upper intestinal fract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of Λe carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, Λe present invention provides a pharmaceutical composition comprising ilodecakin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ilodecakin covalently attached to Λe polypeptide. Preferably, Λe polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iU) a heteropolymer of two or more namrally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synΛetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Uodecakin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of Λe polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide. CW218P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in Λe form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about Λe active agent. In another embodiment, the polypeptide is capable of releasing Λe active agent from Λe composition in a pH-dependent manner.

The invention also provides a method for protecting ilodecakin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ilodecakin to a patient, Λe patient being a human or a non-human animal, comprising administering to Λe patient a composition comprising a polypeptide and an active agent covalently attached to Λe polypeptide. In a prefened embodiment, ilodecakin is released from Λe composition by an enzyme-catalyzed release. In another prefened embodiment, ilodecakin is released in a time-dependent manner based on the pharmacokinetics of Λe enzyme-catalyzed release. In another preferred embodiment, the composition further comprises a microencapsulating agent and ilodecakin is released from the composition by dissolution of the microencapsulating agent. In another preferred embodiment, ilodecakin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, ilodecakin is released from the composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW218P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ilodecakin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) wiΛ a second agent, ilodecakin and a second active agent can be copolymerized in step (c). In anoΛer preferred embodiment, the amino acid is glutamic acid and Λe active agent is released from the glutamic acid as a dimer upon a hydrolysis of Λe polypeptide and wherein Λe active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from Λe group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein Λe active agent is attached to Λe side chain of Λe amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet anoΛer prefeoed embodiment, Λe glutamic acid is replaced by a sjmthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood Λat both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ilodecakin and prevent its digestion in the stomach. In CW218P

addition, the pharmacologic effect can be prolonged by delayed release of ilodecakin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ilodecakin covalently attached to a polypeptide. Preferably, Λe polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more namrally occurring amino acids and one or more synmetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids at have primary, secondary and tertiary stractures. The secondary stracmre of Λe protein is Λe local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on e conformations of the chain deteonine Λe spatial anangement of Λe molecule. The folding of the secondary stracmre and the spatial anangement of Λe side chains constitute the tertiary stracmre.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW218P

and exposing Λem to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds wiΛ water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to Λe overall stability of the folded protein where Λe degree to which ideal packing occurs determines the degree of relative stability of Λe protein. The result of maximum packing is to produce a center of residues or hydrophobic core Λat has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in Λe hydrophobic core of a peptide, it would require energy to unfold the peptide before Λe drug can be released. The unfolding process requires overcoming Λe hydrophobic effect by hydrating Λe amino acids or achieving the melting temperamre of Λe protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neufral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of irreversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated wiΛ the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, Λen the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will CW220P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING IMIGLUCERASE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to imiglucerase, as well as methods for protecting and administering imiglucerase. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Imiglucerase is a known pharmaceutical agent that is used in the treatment of Gaucher disease. Its chemical name is glucosyl-(human placenta isoenzyme protein moiety) 495-L-histidine-ceramidase. It is a recombinant glucocerebrosidase enzyme.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf 4072 - ,„,,,-,-.

CW220P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is ⁴⁰⁷³ CW220P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous . pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW220P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (imiglucerase) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching imiglucerase to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising imiglucerase microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and imiglucerase covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Imiglucerase preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide. CW220P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tab'et, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting imiglucerase from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering imiglucerase to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, imiglucerase is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, imiglucerase is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and imiglucerase is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, imiglucerase is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, imiglucerase is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients. CW220P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching imiglucerase to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, imiglucerase and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize imiglucerase and prevent its digestion in the stomach. In CW220P

addition, the pharmacologic effect can be prolonged by delayed release of imiglucerase. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Imiglucerase is the subject of EP 401362 B (1996), based on priority application

US 289589 (1988), herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises imiglucerase covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW220P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the caoier polypeptide will be CW220P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW220P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen - 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW220P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutaπuc acid and then this conjugate covalently attached to the C-teoninus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW220P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, imiglucerase is covalently attached to the polypeptide via a peptide bond.

The polypeptide canier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW220P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-imiglucerase conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW220P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0 C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW220P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW220P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and imiglucerase covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuning amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuning amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein imiglucerase is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW220P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein imiglucerase is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing imiglucerase from said composition in a pH-dependent manner.

19. A method for protecting imiglucerase from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of imiglucerase from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching imiglucerase to said polypeptide.

21. A method for delivering imiglucerase to a patient comprising administering to said patient a composition comprising: a polypeptide; and imiglucerase covalently attached to said polypeptide.

22. The method of claim 21 wherein imiglucerase is released from said composition by an enzyme-catalyzed release.

17 CW220P

23. The method of claim 21 wherein imiglucerase is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW220P

Abstract

A composition comprising a polypeptide and imiglucerase covalently attached to the polypeptide. Also provided is a method for delivery of imiglucerase to a patient comprising administering to the patient a composition comprising a polypeptide and imiglucerase covalently attached to the polypeptide. Also provided is a method for protecting imiglucerase from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of imiglucerase from a composition comprising covalently attaching it to the polypeptide.

19 CW221P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING IMIPRAMINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to imiprarnine, as well as methods for protecting and administering imiprarnine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a canier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Imipramine is a known phaonaceutical agent that is used in the treatment of depression. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered CW221P

product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations. CW221P

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. CW221P

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (imiprarnine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching imipramine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising imipramine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and imipramine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a CW221P

heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Imiprarnine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting imipramine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering imipramine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, imipramine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, imipramine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed CW221P

release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and imipramine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, imipramine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, imipramine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching imipramine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, imipramine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the CW221P

glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize imipramine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of imipramine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises imipramine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture. CW221P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW221P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW221P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW221P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly fooned free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW221P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

12 CW221P

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of afnino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

13 CW221P

Preferably, the resultant peptide-imipramine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated

14 CW221P

solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-AIkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

15 CW221P

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW221P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and imipramine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein imipramine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW221P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein imipramine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing imipramine from said composition in a pH-dependent manner.

19. A method for protecting imipramine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of imipramine from a composition wherein said composition comprises a polypeptide, said method comprising covalentiy attaching imipramine to said polypeptide.

21. A method for delivering imipramine to a patient comprising administering to said patient a composition comprising: a polypeptide; and imipramine covalently attached to said polypeptide.

22. The method of claim 21 wherein imipramine is released from said composition by an enzyme-catalyzed release. CW221P

23. The method of claim 21 wherein imipramine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

19 CW221P

Abstract

A composition comprising a polypeptide and imipramine covalently attached to the polypeptide. Also provided is a method for delivery of imipramine to a patient comprising administering to the patient a composition comprising a polypeptide and imipramine covalently attached to the polypeptide. Also provided is a method for protecting imipramine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of imipramine from a composition comprising covalently attaching it to the polypeptide.

20 CW222P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING INDINAVIR AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to indinavir, as well as methods for protecting and administering indinavir. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Indinavir is a known pharmaceutical agent that is used in the treatment of HIV infection Its chemical name is 2,3,5-trideoxy-N-[(lS,2R)-2,3-dihydro-2-hydroxy-lH- inden-l-yl]-5-[(2S)-2-[[(l,l-dimethylethyl)amino]carbonyl]-4-(3-pyridinylmethyl)-l- piperazinyl]-2-(phenylmethyl)-D-erythro-pentonamide. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW222P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW222P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the πucroencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soJuble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW222P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (indinavir) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching indinavir to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising indinavir microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and indinavir covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids. CW222P

Indinavir preferably is covalently attached to a side chain, the N-terminus or the C-teoninus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting indinavir from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering indinavir to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, indinavir is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, indinavir is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and indinavir is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, indinavir is released CW222P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, indinavir is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching indinavir to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, indinavir and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW222P

The general applications of this invention to other active phaimaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize indinavir and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of indinavir. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Indinavir is the subject of EP 541168 B (1998), based on priority application US 789508 (1991), and U.S. Patent Number 5,413,999, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises indinavir covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding CW222P

are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW222P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW222P

caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW222P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teoninus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW222P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, indinavir is covalently attached to the polypeptide via the amine groups.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW222P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-indinavir conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW222P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide canier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-teoninus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW222P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutaπuc acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^'

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW222P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and indinavir covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein indinavir is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW222P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet. ^•

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein indinavir is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing indinavir from said composition in a pH-dependent manner.

19. A method for protecting indinavir from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of indinavir from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching indinavir to said polypeptide.

21. A method for delivering indinavir to a patient comprising administering to said patient a composition comprising: a polypeptide; and indinavir covalently attached to said polypeptide.

22. The method of claim 21 wherein indinavir is released from said composition by an enzyme-catalyzed release.

17 CW222P

23. The method of claim 21 wherein indinavir is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW222P

Abstract

A composition comprising a polypeptide and indinavir covalently attached to the polypeptide. Also provided is a method for delivery of indinavir to a patient comprising administering to the patient a composition comprising a polypeptide and indinavir covalently attached to the polypeptide. Also provided is a method for protecting indinavir from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of indinavir from a composition comprising covalently attaching it to the polypeptide.

19 CW223P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING INFLIXIMAB AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to infliximab, as well as methods for protecting and administering infliximab. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Infliximab is a known pharmaceutical agent that is used in the treatment of arthritis and HIV infection. It is a monoclonal antibody targeting tumor necrosis factor alpha. Its chemical name is immunoglobulin G, anti-(human tumour necrosis factor) (human-mouse monoclonal cA2 heavy chain), disulfide with human-mouse monoclonal cA2 light chain, dimer.

The novel pharmaceutical compound. of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase CW223P

markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some CW223P

technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR CW223P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (infliximab) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching infliximab to the N- teoninus, the C-teoninus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Altematively, the present invention provides a pharmaceutical composition comprising infliximab microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and infliximab covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Infliximab preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a preferred embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is CW223P

an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The πucroencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting infliximab from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering infliximab to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, infliximab is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, infliximab is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and infliximab is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, infliximab is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, infliximab is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW223P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching infliximab to a side chain of an amino acid to form an active agent/amino acid complex;

(b) foπriing an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, infliximab and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW223P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize infliximab and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of infliximab. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Infliximab is the subject of U.S. Patent Number yyyyy, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises infliximab covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW223P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW223P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW223P

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW223P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the - terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW223P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, infliximab is covalently attached to the polypeptide via a peptide bond.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW223P

invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-infliximab conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW223P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide canier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW223P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^'

Preparation of Poly [γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW223P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and infliximab covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein infliximab is covalently attached to a side chain, the N-terminus or the C-teoninus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein infliximab is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing infliximab from said composition in a pH-dependent manner.

19. A method for protecting infliximab from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of infliximab from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching infliximab to said polypeptide.

21. A method for delivering infliximab to a patient comprising administering to said patient a composition comprising: a polypeptide; and infliximab covalently attached to said polypeptide.

22. The method of claim 21 wherein infliximab is released from said composition by an enzyme-catalyzed release.

17 CW223P

23. The method of claim 21 wherein infliximab is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW223P

Abstract

A composition comprising a polypeptide and infliximab covalently attached to the polypeptide. Also provided is a method for delivery of infliximab to a patient comprising administering to the patient a composition comprising a polypeptide and infliximab covalently attached to the polypeptide. Also provided is a method for protecting infliximab from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of infliximab from a composition comprising covalently attaching it to the polypeptide.

19 CW224P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING HUMAN INSULIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to human insulin, as well as methods for protecting and administering human insulin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Human insulin is a known pharmaceutical agent that is used in the treatment of diabetes. Insulin human is a biosynthetic or semisynthetic protein that is structurally identical to endogenous insulin secreted by the beta cells of the human pancreas. Although stracturally identical to endogenous human insulin, commercially available insulin human is not extracted from the human pancreas, but is prepared biosynthetically from cultures of genetically modified Escherichia coll or Saccharomyces cerevisiae or semisynthetically by transpeptidation of pork insulin. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical CW224P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW224P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drug delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral - administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW224P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (human insulin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching human insulin to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising human insulin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and human insulin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW224P

Human insulin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting human insulin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering human insulin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, human insulin is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, human insulin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a πucroencapsulating agent and human insulin is released from the composition by dissolution of the microencapsulating agent. In another prefeoed CW224P

embodiment, human insulin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, human insulin is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching human insulin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, human insulin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality. CW224P

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize human insulin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of human insulin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Human insulin is the subject of U.S. Patent Numbers 5,474,978 and 5,514,646, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises human insulin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture. CW224P

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. CW224P

Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the CW224P

kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery: Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's

10 CW224P

molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active

1 1 CW224P

agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, human insulin is covalently attached to the polypeptide via a peptide bond.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW224P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-human insulin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW224P

Compositions ofthe invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

>

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-teoninus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW224P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW224P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW224P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and human insulin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein human insulin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW224P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein human insulin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing human insulin from said composition in a pH-dependent manner.

19. A method for protecting human insulin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of human insulin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching human insulin to said polypeptide.

21. A method for delivering human insulin to a patient comprising administering to said patient a composition comprising: a polypeptide; and human insulin covalently attached to said polypeptide.

22. The method of claim 21 wherein human insulin is released from said composition by an enzyme-catalyzed release.

18 CW224P

23. The method of claim 21 wherein human insulin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

19 CW224P

Abstract

A composition comprising a polypeptide and human insulin covalently attached to the polypeptide. Also provided is a method for delivery of human insulin to a patient comprising administering to the patient a composition comprising a polypeptide and human insulin covalently attached to the polypeptide. Also provided is a method for protecting human insulin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of human insulin from a composition comprising covalently attaching it to the polypeptide.

20 CW225P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING INTERFERON ALFACON-1 AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to interferon alfacon-1, as well as methods for protecting and administering interferon alfacon-1. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Interferon alfacon-1 is a known phaonaceutical agent that is used in the treatment of viral infection and cancer. Its chemical name is interferon alpha 1 (human lymphoblast reduced), N-L-methionyl-22-L-arg-76-L-ala-78-L-asp-79-L-glu-86-L-tyr- 90-L-tyr- 156-L-thr- 157-L-asn- 158-L-leu.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in liέu of an injection or another CW225P

invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW225P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW225P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (interferon alfacon-1) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching interferon alfacon-1 to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising interferon alfacon-1 microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and interferon alfacon-1 covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Interferon alfacon-1 preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide. CW225P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting interferon alfacon-1 from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering interferon alfacon-1 to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, interferon alfacon-1 is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, interferon alfacon-1 is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and interferon alfacon-1 is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, interferon alfacon-1 is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, interferon alfacon-1 is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients. CW225P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching interferon alfacon-1 to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a preferred embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, interferon alfacon-1 and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize interferon alfacon- 1 and prevent its digestion in the stomach. CW225P

In addition, the pharmacologic effect can be prolonged by delayed release of interferon alfacon-1. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Interferon alfacon-1 is the subject of EP 422697 B (1994), based on priority US application 375494 (1982), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises interferon alfacon-1 covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW225P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW225P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW225P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the canier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are preferred because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW225P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide canier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW225P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, interferon alfacon-1 is covalently attached to the polypeptide via a peptide bond.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW225P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-interferon alfacon-1 conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW225P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperamre for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributyiamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW225P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alk l Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW225P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and interferon alfacon-1 covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is- a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein interferon alfacon-1 is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW225P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable

» excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein interferon alfacon-1 is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing interferon alfacon-1 from said composition in a pH-dependent manner.

19. A method for protecting interferon alfacon-1 from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of interferon alfacon-1 from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching interferon alfacon-1 to said polypeptide.

21. A method for delivering interferon alfacon-1 to a patient comprising administering to said patient a composition comprising: a polypeptide; and interferon alfacon-1 covalently attached to said polypeptide.

22. The method of claim 21 wherein interferon alfacon-1 is released from said composition by an enzyme-catalyzed release.

17 CW225P

23. The method of claim 21 wherein interferon alfacon-1 is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW225P

Abstract

A composition comprising a polypeptide and interferon alfacon-1 covalently attached to the polypeptide. Also provided is a method for delivery of interferon alfacon- 1 to a patient comprising administering to the patient a composition comprising a polypeptide and interferon alfacon-1 covalently attached to the polypeptide. Also provided is a method for protecting interferon alfacon-1 from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of interferon alfacon-1 from a composition comprising covalently attaching it to the polypeptide.

19 CW226P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING INTERFERON BETA-1A AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaonaceutical compound that comprises a polypeptide that is preferably covalently attached to interferon beta- la, as well as methods for protecting and administering interferon beta- la. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Interferon beta- la is a known phaonaceutical agent that is used in the treatment of multiple sclerosis, viral infection and cancer. It is 145258-61-3 human fibroblast protein moiety 74899-73-3 pre-(human fibroblast protein moiety reduced) 74899-71-1 human fibroblast protein moiety reduced. Biogen was awarded European patent number 41313 for the production of interferon beta through recombinant technology. The patent covered recombinant DNA molecules, transformed hosts and methods for producing recombinant interferon beta proteins.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW226P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW226P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW226P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (interferon beta- la) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching interferon beta- la to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising interferon beta- la microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and interferon beta- la covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Interferon beta- la preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW226P

the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting interferon beta- la from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering interferon beta- la to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, interferon beta- la is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, interferon beta- la is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and interferon beta- la is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, interferon beta- la is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, interferon beta- la is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the CW226P

polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching interferon beta- la to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, interferon beta- la and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW226P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize interferon beta- la and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of interferon beta- la. Furthermore, active agents can be combined to produce synergistic effects.

Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises interferon beta- la covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW226P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW226P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW226P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by. any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW226P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-teoninus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teoninus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW226P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, interferon beta- la is covalently attached to the polypeptide via a peptide bond.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW226P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-interferon beta- la conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW226P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide canier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide canier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concenttated hydrochloric acid can be prepared and heated for

14 CW226P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl GIutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW226P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and interferon beta- la covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is- a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein interferon beta- la is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW226P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein interferon beta-la is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing interferon beta- la from said composition in a pH-dependent manner.

19. A method for protecting interferon beta- la from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of interferon beta- la from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching interferon beta- la to said polypeptide.

21. A method for delivering interferon beta- la to a patient comprising administering to said patient a composition comprising: a polypeptide; and interferon beta- la covalently attached to said polypeptide.

22. The method of claim 21 wherein interferon beta- la is released from said composition by an enzyme-catalyzed release.

17 CW226P

23. The method of claim 21 wherein interferon beta-la is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW226P

Abstract

A composition comprising a polypeptide and interferon beta- la covalently attached to the polypeptide. Also provided is a method for delivery of interferon beta- la to a patient comprising administering to the patient a composition comprising a polypeptide and interferon beta- la covalently attached to the polypeptide. Also provided is a method for protecting interferon beta- la from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of interferon beta- la from a composition comprising covalently attaching it to the polypeptide.

19 CW227P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING INTERLEUKIN-2 AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to interleukin-2, as well as methods for protecting and administering interleukin-2. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Interleukin-2 is a known pharmaceutical agent that is used in the treatment of renal cell carcinoma. IL-2 promotes proliferation, differentiation, and recruitment of T and B cells, natural killer (NK) cells, and thymocytes; IL-2 also causes cytolytic activity in a subset of lymphocytes and subsequent interactions between the immune system and malignant cells; IL-2 can stimulate lymphokine-activated killer (LAK) cells and tumor- infiltrating lymphocytes (TIL) cells. LAK cells (which are derived from lymphocytes from a patient and incubated in TL-2) have the ability to lyse cells which are resistant to NK cells.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW227P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW227P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW227P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (interleukin-2) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching interleukin-2 to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising interleukin-2 microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and interleukin-2 covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Interleukin-2 preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW227P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a " microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting interleukin-2 from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering interleukin-2 to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, interleukin-2 is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, interleukin-2 is . released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and interleukin-2 is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, interleukin-2 is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, interleukin-2 is released from the CW227P

composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching interleukin-2 to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, interleukin-2 and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW227P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize interleukin-2 and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of interleukin-2. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises interleukin-2 covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW227P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein intenor and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW227P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW227P

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW227P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit fooning a new peptide bond. If the active agent is an alcohol, then either the C-teoninus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drug as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutaπuc acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW227P

maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, interleukin-2 is covalently attached to the polypeptide via a peptide bond.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW227P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefened when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-interleukin-2 conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW227P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW227P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW227P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and interleukin-2 covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein interleukin-2 is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW227P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein interleukin-2 is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing interleukin-2 from said composition in a pH-dependent manner.

19. A method for protecting interleukin-2 from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of interleukin-2 from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching interleukin-2 to said polypeptide.

21. A method for delivering interleukin-2 to a patient comprising administering to said patient a composition comprising: a polypeptide; and interleukin-2 covalently attached to said polypeptide.

22. The method of claim 21 wherein interleukin-2 is released from said composition by an enzyme-catalyzed release.

17 CW227P

23. The method of claim 21 wherein interleukin-2 is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW227P

Abstract

A composition comprising a polypeptide and interleukin-2 covalently attached to the polypeptide. Also provided is a method for delivery of interleukin-2 to a patient comprising administering to the patient a composition comprising a polypeptide and interleukin-2 covalently attached to the polypeptide. Also provided is a method for protecting interleukin-2 from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of interleukin-2 from a composition comprising covalently attaching it to the polypeptide.

19 CW228P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING IODIXANOL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to iodixanol, as well as methods for protecting and administering iodixanol. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Iodixanol is a known pharmaceutical agent that is used as a contrast medium for medical imaging. Its chemical name is 5,5'-[(2-hydroxy-l,3- propanediyl)bis(acetylamino)]bis[N,N'-bis(2,3-dih ydroxypropyl)-2,4,6-triodo-l,3- benzenedicarboxamide]. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability CW228P

of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified CW228P

amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the πucroencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that CW228P

incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (iodixanol) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching iodixanol to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising iodixanol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and iodixanol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a CW228P

heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Iodixanol preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting iodixanol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering iodixanol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, iodixanol is released from the composition by CW228P

an enzyme-catalyzed release. In another prefeoed embodiment, iodixanol is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and iodixanol is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, iodixanol is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, iodixanol is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching iodixanol to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, iodixanol and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the CW228P

glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize iodixanol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of iodixanol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Iodixanol is the subject of U.S. Patent Number 5,349,085, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises iodixanol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and CW228P

at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain CW228P

length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

10 CW228P

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to

1 1 CW228P

poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

12 CW228P

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, iodixanol is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the

13 CW228P

mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-iodixanol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation „ -

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

14 CW228P

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

15 CW228P

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW228P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and iodixanol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

•^"\

8. The composition of claim 1 wherein iodixanol is covalently attached to a side chain, the N-teoninus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW228P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein iodixanol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing iodixanol from said composition in a pH-dependent manner.

19. A method for protecting iodixanol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of iodixanol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching iodixanol to said polypeptide.

21. A method for delivering iodixanol to a patient comprising administering to said patient a composition comprising: a polypeptide; and iodixanol covalently attached to said polypeptide.

22. The method of claim 21 wherein iodixanol is released from said composition by an enzyme-catalyzed release.

18 CW228P

23. The method of claim 21 wherein iodixanol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and iodixanol covalently attached to the polypeptide. Also provided is a method for delivery of iodixanol to a patient comprising administering to the patient a composition comprising a polypeptide and iodixanol covalently attached to the polypeptide. Also provided is a method for protecting iodixanol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of iodixanol from a composition comprising covalently attaching it to the polypeptide.

19 CW229P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING IOPROMIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to iopromide, as well as methods for protecting and administering iopromide. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Iopromide is a known pharmaceutical agent that is used as an X-ray contrast medium. Its chemical name is N,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-5-[(2- methoxyacetyl)amino]-N-methyl-l,3-benzenedicarboxamide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical CW229P

compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility arid perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet fo nulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW229P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW229P

diffusion rates and pharmacokinetics. High molecular weight caniers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (iopromide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching iopromide to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising iopromide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and iopromide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids. CW229P

Iopromide preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting iopromide from degradation comprising cov,alently attaching it to a polypeptide.

The invention also provides a method for delivering iopromide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, iopromide is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, iopromide is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and iopromide is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, iopromide is released CW229P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, iopromide is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching iopromide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, iopromide and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW229P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize iopromide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of iopromide. Furthermore, active agents can be combined to produce synergistic ffects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Iopromide is the subject of U.S. Patent Number 4,364,921, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises iopromide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW229P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW229P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW229P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW229P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit fooning a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teoninus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly fooned free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

11 CW229P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, iopromide is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW229P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-iopromide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW229P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW229P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly [γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW229P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and iopromide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein iopromide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW229P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein iopromide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing iopromide from said composition in a pH-dependent manner.

" 19. A method for protecting iopromide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of iopromide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching iopromide to said polypeptide.

21. A method for delivering iopromide to a patient comprising administering to said patient a composition comprising: a polypeptide; and iopromide covalently attached to said polypeptide.

22. The method of claim 21 wherein iopromide is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein iopromide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

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Abstract

A composition comprising a polypeptide and iopromide covalently attached to the polypeptide. Also provided is a method for delivery of iopromide to a patient comprising administering to the patient a composition comprising a polypeptide and iopromide covalently attached to the polypeptide. Also provided is a method for protecting iopromide from degradation comprising covalently attaching it to a polypeptide. Alsc provided is a method for controlling release of iopromide from a composition comprising covalently attaching it to the polypeptide.

19 CW230P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING IOXAGLATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ioxaglate, as well as methods for protecting and administering ioxaglate. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ioxaglate is a known pharmaceutical agent that is used as a radiopaque contrast aide. It is usually used as a combination of Ioxaglate meglumine and Ioxaglate sodium. Both units can be attached to a polypeptide caoier.

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW230P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW230P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW230P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ioxaglate) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching ioxaglate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ioxaglate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ioxaglate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ioxaglate preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide. CW230P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tabiet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ioxaglate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ioxaglate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, ioxaglate is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, ioxaglate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ioxaglate is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, ioxaglate is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ioxaglate is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW230P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ioxaglate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ioxaglate and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ioxaglate and prevent its digestion in the stomach. In addition, CW230P

the pharmacologic effect can be prolonged by delayed release of ioxaglate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ioxaglate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein, stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW230P

and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, carrbe selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will CW230P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the CW230P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order

10 CW230P

to achieve a stable peptide linked active agent. In both, the C- and N-teoninus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teoninus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

1 1 CW230P

preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ioxaglate is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide caoier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

12 CW230P

intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ioxaglate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

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The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

14 CW230P

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW230P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ioxaglate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ioxaglate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW230P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ioxaglate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ioxaglate from said composition in a pH-dependent manner.

19. A method for protecting ioxaglate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ioxaglate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ioxaglate to said polypeptide.

21. A method for delivering ioxaglate to a patient comprising administering to said patient a composition comprising: a polypeptide; and ioxaglate covalently attached to said polypeptide.

22. The method of claim 21 wherein ioxaglate is released from said composition by an enzyme-catalyzed release.

17 CW230P

23. The method of claim 21 wherein ioxaglate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW230P

Abstract

A composition comprising a polypeptide and ioxaglate covalently attached to the polypeptide. Also provided is a method for delivery of ioxaglate to a patient comprising administering to the patient a composition comprising a polypeptide and ioxaglate covalently attached to the polypeptide. Also provided is a method for protecting ioxaglate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ioxaglate from a composition comprising covalently attaching it to the polypeptide.

19 CW231P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING IPRATROPIUM AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ipratropium, as well as methods for protecting and administering ipratropium. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ipratropium is used as a bronchodilator for the long-term symptomatic treatment of reversible bronchospasm associated with chronic obstractive pulmonary disease (COPD). Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW231P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW231P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet/another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW231P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ipratropium) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ipratropium to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising ipratropium microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ipratropium covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ipratropium preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW231P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ipratropium from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ipratropium to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, ipratropium is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, ipratropium is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ipratropium is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, ipratropium is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ipratropium is released from the CW231P

composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ipratropium to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ipratropium and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW231P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ipratropium and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ipratropium. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ipratropium covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW231P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW231P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW231P

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW231P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW231P

maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ipratropium is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW231P

invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ipratropium conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW231P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW231P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^'

Preparation of Poly [γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW231P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ipratropium covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ipratropium is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said πucroencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW231P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ipratropium is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ipratropium from said composition in a pH-dependent manner.

19. A method for protecting ipratropium from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ipratropium from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ipratropium to said polypeptide.

21. A method for delivering ipratropium to a patient comprising administering to said patient a composition comprising: a polypeptide; and ipratropium covalently attached to said polypeptide.

22. The method of claim 21 wherein ipratropium is released from said composition by an enzyme-catalyzed release.

17 CW231P

23. The method of claim 21 wherein ipratropium is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW231P

Abstract

A composition comprising a polypeptide and ipratropium covalently attached to the polypeptide. Also provided is a method for delivery of ipratropium to a patient comprising administering to the patient a composition comprising a polypeptide and ipratropium covalently attached to the polypeptide. Also provided is a method for protecting ipratropium from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ipratropium from a composition comprising covalently attaching it to the polypeptide.

19 CW232P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING IRBESARTAN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to irbesartan, as well as methods for protecting and administering irbesartan. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Irbesartan is a known pharmaceutical agent that is used in the treatment of hypertension. Its chemical name is 2-butyl-3-[[2'-(lH-tetrazol-5-yl)[l,l'-biphenyl]-4- yl]methyl]-l,3-diazaspiro[4.4]non-l-en-4-one. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW232P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid πucrospheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW232P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW232P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (irbesartan) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching irbesartan to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising irbesartan microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and irbesartan covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occuning amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Irbesartan preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW232P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting irbesartan from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering irbesartan to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, irbesartan is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, irbesartan is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and irbesartan is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, irbesartan is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, irbesartan is released from the composition in a sustained release. CW232P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching irbesartan to a side chain of an amino acid to form an active agent/amino acid complex; (b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, irbesartan and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW232P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize irbesartan and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of irbesartan. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Irbesartan is the subject of U.S. Patent Number 5,270,317, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises irbesartan covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW232P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW232P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the canier polypeptide. Another, significant advantage of the invention is that the kinetics of CW232P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW232P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-teoninus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW232P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide canier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW232P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-irbesartan conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW232P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW232P Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW232P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and irbesartan covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein irbesartan is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW232P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein irbesartan is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing irbesartan from said composition in a pH-dependent manner.

19. A method for protecting irbesartan from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of irbesartan from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching irbesartan to said polypeptide.

21. A method for delivering irbesartan to a patient comprising administering to said patient a composition comprising: a polypeptide; and irbesartan covalently attached to said polypeptide.

22. The method of claim 21 wherein irbesartan is released from said composition by an enzyme-catalyzed release.

17 CW232P

23. The method of claim 21 wherein irbesartan is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW232P

Abstract ^"

A composition comprising a polypeptide and irbesartan covalently attached to the polypeptide. Also provided is a method for delivery of irbesartan to a patient comprising administering to the patient a composition comprising a polypeptide and irbesartan covalently attached to the polypeptide. Also provided is a method for protecting irbesartan from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of irbesartan from a composition comprising covalently attaching it to the polypeptide.

19 CW233P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING IRINOTECAN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to irinotecan, as well as methods for protecting and administering irinotecan. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Irinotecan is a known pharmaceutical agent that is used in the treatment of cancer. Its chemical name is [l,4'-bipiperidine]-l '-carboxylic acid (S)-4,l l-diethyl-3,4,12,14- tetrahydro-4-hydroxy-3, 14-dioxo- lH-pyrano[3',4':6,7]indolizino[ 1 ,2-b]quinolin-9-yl ester. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical CW233P

compound may contain one or more of the following: another active phaonaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several CW233P

shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable CW233P

diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (irinotecan) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching irinotecan to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising irinotecan microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and irinotecan covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids. CW233P

Irinotecan preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- te ninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting irinotecan from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering irinotecan to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, irinotecan is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, irinotecan is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and irinotecan is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, irinotecan is released CW233P

from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, irinotecan is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of: (a) attaching irinotecan to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, irinotecan and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. CW233P

The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize irinotecan and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of irinotecan. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Irinotecan is the subject of U.S. Patent Number 4,604,463, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises irinotecan covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW233P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW233P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW233P

caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW233P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covaleutly attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutaπuc acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW233P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, irinotecan is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW233P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-irinotecan conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW233P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

AIcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-teoninus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW233P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW233P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and irinotecan covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein irinotecan is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW233P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein irinotecan is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing irinotecan from said composition in a pH-dependent manner.

19. A method for protecting irinotecan from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of irinotecan from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching irinotecan to said polypeptide.

21. A method for delivering irinotecan to a patient comprising administering to said patient a composition comprising: a polypeptide; and irinotecan covalently attached to said polypeptide.

22. The method of claim 21 wherein irinotecan is released from said composition by an enzyme-catalyzed release.

17 CW233P

23. The method of claim 21 wherein irinotecan is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW233P

Abstract

A composition comprising a polypeptide and irinotecan covalently attached to the polypeptide. Also provided is a method for delivery of irinotecan to a patient comprising administering to the patient a composition comprising a polypeptide and irinotecan covalently attached to the polypeptide. Also provided is a method for protecting irinotecan from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of irinotecan from a composition comprising covalently attaching it to the polypeptide.

19 CW234P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ISOSORBIDE DINITRATE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to isosorbide dinitrate, as well as methods for protecting and administering isosorbide dinitrate. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Isosorbide dinitrate is a known phaimaceutical agent that is used in the treatment of angina. It is made up of the organic nitrates and nitrites are esters of nitrous or nitric acid, primarily amyl nitrite.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement, of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW234P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW234P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW234P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (isosorbide dinitrate) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching isosorbide dinitrate to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising isosorbide dinitrate microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and isosorbide dinitrate covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Isosorbide dinitrate preferably is covalently attached to a side chain, the N- terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-terminus of the polypeptide. CW234P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting isosorbide dinitrate from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering isosorbide dinitrate to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, isosorbide dinitrate is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, isosorbide dinitrate is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and isosorbide dinitrate is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, isosorbide dinitrate is released from the composition by a pH- dependent unfolding of the polypeptide. In another prefeoed embodiment, isosorbide dinitrate is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients. CW234P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching isosorbide dinitrate to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, isosorbide dinitrate and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another preferred embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize isosorbide dinitrate and prevent its digestion in the stomach. CW234P

In addition, the pharmacologic effect can be prolonged by delayed release of isosorbide dinitrate. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises isosorbide dinitrate covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW234P

and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will CW234P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the CW234P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order

10 CW234P

to achieve a stable peptide linked active agent. In both, the C- and N-teoninus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-teoninus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

11 CW234P

preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, isosorbide dinitrate is covalently attached to the polypeptide via the nitrite group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

12 CW234P

intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-isosorbide dinitrate conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

13 CW234P

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

14 CW234P

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW234P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and isosorbide dinitrate covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein isosorbide dinitrate is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW234P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein isosorbide dinitrate is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing isosorbide dinitrate from said composition in a pH-dependent manner.

19. A method for protecting isosorbide dinitrate from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of isosorbide dinitrate from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching isosorbide dinitrate to said polypeptide.

21. A method for delivering isosorbide dinitrate to a patient comprising administering to said patient a composition comprising: a polypeptide; and isosorbide dinitrate covalently attached to said polypeptide.

22. The method of claim 21 wherein isosorbide dinitrate is released from said composition by an enzyme-catalyzed release.

17 CW234P

23. The method of claim 21 wherein isosorbide dinitrate is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW234P

Abstract

A composition comprising a polypeptide and isosorbide dinitrate covalently attached to the polypeptide. Also provided is a method for delivery of isosorbide dinitrate to a patient comprising administering to the patient a composition comprising a polypeptide and isosorbide dinitrate covalently attached to the polypeptide. Also provided is a method for protecting isosorbide dinitrate from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of isosorbide dinitrate from a composition comprising covalently attaching it to the polypeptide.

19 CW235P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ISOTRETINOIN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to isotretinoin, as well as methods for protecting and administering isotretinoin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a caoier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Isotretinoin is a known phaimaceutical agent that is used in the treatment of acne. Its stracture is:

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken CW235P

under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent CW235P

in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon- specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration. -

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR CW235P

application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (isotretinoin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching isotretinoin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also referred to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising isotretinoin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and isotretinoin covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Isotretinoin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefened embodiment, the active agent is CW235P

an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefened embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting isotretinoin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering isotretinoin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, isotretinoin is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, isotretinoin is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and isotretinoin is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, isotretinoin is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefened embodiment, isotretinoin is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be CW235P

microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching isotretinoin to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, isotretinoin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW235P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize isotretinoin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of isotretinoin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises isotretinoin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW235P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these, forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW235P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neuttal in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW235P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW235P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW235P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drug dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, isotretinoin is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW235P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-isotretinoin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW235P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW235P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW235P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and isotretinoin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein isotretinoin is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW235P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein isotretinoin is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing isotretinoin from said composition in a pH-dependent manner.

19. A method for protecting isotretinoin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of isotretinoin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching isotretinoin to said polypeptide.

21. A method for delivering isotretinoin to a patient comprising administering to said patient a composition comprising: a polypeptide; and isotretinoin covalently attached to said polypeptide.

22. The method of claim 21 wherein isotretinoin is released from said composition by an enzyme-catalyzed release.

17 CW235P

23. The method of claim 21 wherein isotretinoin is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW235P

Abstract

A composition comprising a polypeptide and isotretinoin covalently attached to the polypeptide. Also provided is a method for delivery of isotretinoin to a patient comprising administering to the patient a composition comprising a polypeptide and isotretinoin covalently attached to the polypeptide. Also provided is a method for protecting isotretinoin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of isotretinoin from a composition comprising covalently attaching it to the polypeptide.

19 CW236P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ISRADIPINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to isradipine, as well as methods for protecting and administering isradipine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a caoier compound that enhances the usefulness of the phaonaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Isradipine is a known pharmaceutical agent that is used in the treatment of hypertension. Its chemical name is 4-(4-benzofurazanyl)-l,4-dihydro-2,6-dimethyl-3,5- pyridinedicarboxylic acid methyl 1-methylethyl ester. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor. CW236P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW236P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW236P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (isradipine) to a polymer of peptides or amino acids. The invention' is distinguished from the above-mentioned technologies by virtue of covalently attaching isradipine to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising isradipine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and isradipine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Isradipine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW236P

carboxylic acid and is covalently attached to the N-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting isradipine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering isradipine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, isradipine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, isradipine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and isradipine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, isradipine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, isradipine is released from the composition in a sustained release. CW236P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching isradipine to a side chain of an amino acid to form an active agent amino acid complex; (b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, isradipine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW236P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize isradipine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of isradipine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Isradipine is the subject of EP 150 B (1981) and UK 2037766 B (1983), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises isradipine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW236P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW236P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW236P

carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW236P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide canier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW236P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides.

12 CW236P

Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-isradipine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

13 CW236P

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide canier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stined for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N-

14 CW236P

hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C.

The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather,

15 CW236P

various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW236P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and isradipine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein isradipine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

17 CW236P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein isradipine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing isradipine from said composition in a pH-dependent manner.

19. A method for protecting isradipine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of isradipine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching isradipine to said polypeptide.

21. A method for delivering isradipine to a patient comprising administering to said patient a composition comprising: a polypeptide; and isradipine covalently attached to said polypeptide.

22. The method of claim 21 wherein isradipine is released from said composition by an enzyme-catalyzed release.

18 CW236P

23. The method of claim 21 wherein isradipine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

19 CW236P

Abstract

A composition comprising a polypeptide and isradipine covalently attached to the polypeptide. Also provided is a method for delivery of isradipine to a patient comprising administering to the patient a composition comprising a polypeptide and isradipine covalently attached to the polypeptide. Also provided is a method for protecting isradipine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of isradipine from a composition comprising covalently attaching it to the polypeptide.

20 CW237P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ITASETRON AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to itasetron, as well as methods for protecting and administering itasetron. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Itasetron is a known pharmaceutical agent that is used in the treatment of emesis and anxiety. Its chemical name is endo-2,3-dihydro-N-(8-methyl-8-azabicyclo[3.2.1]oct- 3-yl)-2-oxo-lH-benzimidazole-l-carboxamide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW237P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW237P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW237P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (itasetron) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching itasetron to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising itasetron microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and itasetron covalently attached to the polypeptide. Preferably, the polypeptide is (i)_.an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Itasetron preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW237P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting itasetron from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering itasetron to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, itasetron is released from the composition by an enzyme-catalyzed release. In another preferred embodiment, itasetron is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and itasetron is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, itasetron is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, itasetron is released from the composition in a sustained release. CW237P

In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching itasetron to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, itasetron and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is CW237P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize itasetron and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of itasetron. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Itasetron is the subject of EP 309423 B (1994), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises itasetron covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW237P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperamre requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW237P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of CW237P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) ^' 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW237P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefened point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teoninus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

1 1 CW237P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, itasetron is covalently attached to the polypeptide via the amine group.

The polypeptide canier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW237P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-itasetron conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW237P

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW237P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^'

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW237P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and itasetron covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein itasetron is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW237P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein itasetron is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing itasetron from said composition in a pH-dependent manner.

19. A method for protecting itasetron from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of itasetron from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching itasetron to said polypeptide.

21. A method for delivering itasetron to a patient comprising administering to said patient a composition comprising: a polypeptide; and itasetron covalently attached to said polypeptide.

22. The method of claim 21 wherein itasetron is released from said composition by an enzyme-catalyzed release.

17 CW237P

23. The method of claim 21 wherein itasetron is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW237P

Abstract

A composition comprising a polypeptide and itasetron covalently attached to the polypeptide. Also provided is a method for delivery of itasetron to a patient comprising administering to the patient a composition comprising a polypeptide and itasetron covalently attached to the polypeptide. Also provided is a method for protecting itasetron from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of itasetron from a composition comprising covalently attaching it to the polypeptide.

19 CW238P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ITRACONAZOLE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to itraconazole, as well as methods for protecting and administering itraconazole. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Itraconazole is a known pharmaceutical agent that is used in the treatment of mycosis. Its chemical name is 4-[4-[4-[4-[[2-(2,4-dichlorophenyl)-2-[(lH-l,2,4-triazol- l-yl)methyl]-l,3-dioxolan-4-yl]methoxy]phenyl]-l-piperazinyl]phenyl]-2,4-dihydro-2- (l-methylpropyl)-3H-l,2,4-triazol-3-one. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW238P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW238P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW238P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (itraconazole) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching itraconazole to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising itraconazole microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and itraconazole covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Itraconazole preferably is covalently attached to a side chain, the N-teoninus or the C-teoninus of the polypeptide. In a prefeoed embodiment, the active agent is a CW238P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting itraconazole from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering itraconazole to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, itraconazole is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, itraconazole is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and itraconazole is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, itraconazole is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, itraconazole is released from the CW238P

composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching itraconazole to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, itraconazole and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW238P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize itraconazole and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of itraconazole. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Itraconazole is the subject of U.S. Patent Numbers 4,267,179, 4,727,064, and

5,707,975, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises itraconazole covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary structure of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW238P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, confϊgurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW238P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of CW238P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW238P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-teoninus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using CW238P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW238P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-itraconazole conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW238P

AcidVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW238P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW238P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and itraconazole covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein itraconazole is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW238P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein itraconazole is conformationally protected by folding of said polypeptide about said active agent.

• 18. The composition of claim 1 wherein said polypeptide is capable of releasing itraconazole from said composition in a pH-dependent manner.

19. A method for protecting itraconazole from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of itraconazole from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching itraconazole to said polypeptide.

21. A method for delivering itraconazole to a patient comprising administering to said patient a composition comprising: a polypeptide; and itraconazole covalently attached to said polypeptide.

22. The method of claim 21 wherein itraconazole is released from said composition by an enzyme-catalyzed release.

17 CW238P

23. The method of claim 21 wherein itraconazole is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW238P

Abstract

A composition comprising a polypeptide and itraconazole covalently attached to the polypeptide. Also provided is a method for delivery of itraconazole to a patient comprising administering to the patient a composition comprising a polypeptide and itraconazole covalently attached to the polypeptide. Also provided is a method for protecting itraconazole from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of itraconazole from a composition comprising covalently attaching it to the polypeptide.

19 CW239P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING KETOCONAZOLE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ketoconazole, as well as methods for protecting and administering ketoconazole. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ketoconazole is used in the treatment of blastomycosis, candidal infections (i.e., oropharyngeal and/or esophageal candidiasis, vulvovaginal candidiasis, candiduria, chronic mucocutaneous candidiasis), chromomycosis (chromoblastomycosis), coccidioidomycosis, histoplasmosis, and paracoccidioidomycosis. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW239P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW239P

reproducibility. In addition, encapsulated drugs rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW239P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ketoconazole) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ketoconazole to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising ketoconazole microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ketoconazole covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuning amino acids and one or more synthetic amino acids.

Ketoconazole preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefened embodiment, the active agent is a CW239P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ketoconazole from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ketoconazole to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, ketoconazole is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, ketoconazole is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ketoconazole is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, ketoconazole is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ketoconazole is released from the CW239P

composition in a sustained release. In yet another prefened embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ketoconazole to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ketoconazole and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is CW239P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ketoconazole and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ketoconazole. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced: The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ketoconazole covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuning amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW239P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are ι

"pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neuttal pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW239P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW239P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW239P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW239P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide canier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW239P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ketoconazole conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW239P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW239P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-AlkyI Glutamate]

γ-Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW239P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ketoconazole covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ketoconazole is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW239P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ketoconazole is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ketoconazole from said composition in a pH-dependent manner.

19. A method for protecting ketoconazole from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ketoconazole from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ketoconazole to said polypeptide.

21. A method for delivering ketoconazole to a patient comprising administering to said patient a composition comprising: a polypeptide; and ketoconazole covalently attached to said polypeptide.

22. The method of claim 21 wherein ketoconazole is released from said composition by an enzyme-catalyzed release.

17 CW239P

23. The method of claim 21 wherein ketoconazole is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW239P

Abstract

A composition comprising a polypeptide and ketoconazole covalently attached to the polypeptide. Also provided is a method for delivery of ketoconazole to a patient comprising administering to the patient a composition comprising a polypeptide and ketoconazole covalently attached to the polypeptide. Also provided is a method for protecting ketoconazole from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ketoconazole from a composition comprising covalently attaching it to the polypeptide.

19 CW240P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING KETOPROFEN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ketoprofen, as well as methods for protecting and administering ketoprofen. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ketoprofen is a known phaonaceutical agent that is used in the treatment of arthritis and pain. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaimaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW240P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW240P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW240P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(ketoprofen) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching ketoprofen to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising ketoprofen microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ketoprofen covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ketoprofen preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW240P

the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ketoprofen from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ketoprofen to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, ketoprofen is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, ketoprofen is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ketoprofen is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, ketoprofen is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ketoprofen is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the CW240P

composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ketoprofen to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ketoprofen and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW240P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ketoprofen and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ketoprofen. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ketoprofen covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW240P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW240P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW240P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

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The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit fooning a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-teoninus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutaπuc acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

1 1 CW240P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, poljtyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ketoprofen is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide caoier can be prepared using conventional techniques. A prefened technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

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There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ketoprofen conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW240P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW240P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW240P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and ketoprofen covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ketoprofen is covalently attached to a side chain, the N-terminus or the C-terminus of said poljφeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW240P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ketoprofen is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ketoprofen from said composition in a pH-dependent manner.

19. A method for protecting ketoprofen from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ketoprofen from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ketoprofen to said polypeptide.

21. A method for delivering ketoprofen to a patient comprising administering to said patient a composition comprising: a polypeptide; and ketoprofen covalently attached to said polypeptide.

22. The method of claim 21 wherein ketoprofen is released from said composition by an enzyme-catalyzed release.

17 CW240P

23. The method of claim 21 wherein ketoprofen is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW240P

Abstract

A composition comprising a polypeptide and ketoprofen covalently attached to the polypeptide. Also provided is a method for delivery of ketoprofen to a patient comprising administering to the patient a composition comprising a polypeptide and ketoprofen covalently attached to the polypeptide. Also provided is a method for protecting ketoprofen from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ketoprofen from a composition comprising covalently attaching it to the polypeptide.

19 CW241P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING KETOROLAC AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaonaceutical compound that comprises a polypeptide that is preferably covalently attached to ketorolac, as well as methods for protecting and administering ketorolac. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ketorolac is a known pharmaceutical agent that is used in the treatment of pain. Its chemical name is (+,-)-5-benzoyl-2,3-dihydro-lH-pyoolizine-l-carboxylic acid. Its structure is:

The novel phaonaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW241P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW241P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drug delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW241P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ketorolac) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching ketorolac to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising ketorolac microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ketorolac covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ketorolac preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefened embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW241P

the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ketorolac from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ketorolac to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, ketorolac is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, ketorolac is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ketorolac is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, ketorolac is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ketorolac is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW241P

controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ketorolac to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ketorolac and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutaπuc acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW241P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ketorolac and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of ketorolac. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Ketorolac is the subject of GB 1554057 (1979), based on priority application US 704909 (1976), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises ketorolac covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain deteonine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW241P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW241P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW241P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW241P

groups of decaglutamic acid, for instance, a drug with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

11 CW241P

maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ketorolac is covalently attached to the polypeptide via the carboxylic acid group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW241P

invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport, system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ketorolac conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

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Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW241P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW241P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and ketorolac covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ketorolac is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW241P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ketorolac is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ketorolac from said composition in a pH-dependent manner.

19. A method for protecting ketorolac from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ketorolac from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ketorolac to said polypeptide.

21. A method for delivering ketorolac to a patient comprising administering to said patient a composition comprising: a polypeptide; and ketorolac covalently attached to said polypeptide.

22. The method of claim 21 wherein ketorolac is released from said composition by an enzyme-catalyzed release.

17 CW241P

23. The method of claim 21 wherein ketorolac is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW241P

Abstract

A composition comprising a polypeptide and ketorolac covalently attached to the polypeptide. Also provided is a method for delivery of ketorolac to a patient comprising administering to the patient a composition comprising a polypeptide and ketorolac covalently attached to the polypeptide. Also provided is a method for protecting ketorolac from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ketorolac from a composition comprising covalently attaching it to the polypeptide.

19 CW242P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING KETOTIFEN AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to ketotifen, as well as methods for protecting and administering ketotifen. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a caoier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Ketotifen is a known pharmaceutical agent that is used in the treatment of allergic conjunctivitis.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf CW242P

life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or Λermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is CW242P

unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drug and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns. CW242P

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (ketotifen) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching ketotifen to the N-teoninus, the C-teoninus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising ketotifen microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and ketotifen covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Ketotifen preferably is covalently attached to a side chain, the N-teoninus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teiminus of the polypeptide. CW242P

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting ketotifen from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering ketotifen to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, ketotifen is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, ketotifen is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and ketotifen is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, ketotifen is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, ketotifen is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW242P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching ketotifen to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, ketotifen and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize ketotifen and prevent its digestion in the stomach. In addition, CW242P

the pharmacologic effect can be prolonged by delayed release of ketotifen. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises ketotifen covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior CW242P

and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will CW242P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drag for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the CW242P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dexttan. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order

10 CW242P

to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly fooned free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

u CW242P

preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, ketotifen is covalently attached to the polypeptide via a hydroxyl, amine or carboxylic acid group or, alternatively, via an artificial linker..

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

12 CW242P

intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-ketotifen conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

13 CW242P

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

14 CW242P

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW242P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and ketotifen covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein ketotifen is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein ketotifen is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing ketotifen from said composition in a pH-dependent manner.

19. A method for protecting ketotifen from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of ketotifen from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching ketotifen to said polypeptide.

21. A method for delivering ketotifen to a patient comprising administering to said patient a composition comprising: a polypeptide; and ketotifen covalently attached to said polypeptide.

22. The method of claim 21 wherein ketotifen is released from said composition by an enzyme-catalyzed release.

17 CW242P

23. The method of claim 21 wherein ketotifen is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW242P

Abstract

A composition comprising a polypeptide and ketotifen covalently attached to the polypeptide. Also provided is a method for delivery of ketotifen to a patient comprising administering to the patient a composition comprising a polypeptide and ketotifen covalently attached to the polypeptide. Also provided is a method for protecting ketotifen from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of ketotifen from a composition comprising covalently attaching it to the polypeptide.

19 CW243P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING LABETALOL AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to labetalol, as well as methods for protecting and administering labetalol. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaimaceutical agent that is both well studied and occupies a known segment of the phaimaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Labetalol is a known pharmaceutical agent that is used in the treatment of hypertension. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaimaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW243P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW243P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW243P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (labetalol) to a polymer of peptides or amino acids. The invention is distinguished from the above- mentioned technologies by virtue of covalently attaching labetalol to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising labetalol microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and labetalol covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Labetalol preferably is covalently attached to a side chain, the N-terminus or the

C-teoninus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW243P

the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. "_ he microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting labetalol from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering labetalol to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, labetalol is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, labetalol is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and labetalol is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, labetalol is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, labetalol is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW243P

controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching labetalol to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefened embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, labetalol and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefened embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefened embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW243P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize labetalol and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of labetalol. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises labetalol covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state. CW243P

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these orces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be CW243P

enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate CW243P

weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileurn. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

10 CW243P

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

11 CW243P

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, labetalol is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

12 CW243P

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-labetalol conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to

0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product

13 CW243P

precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for

14 CW243P

several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

¹ The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW243P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and labetalol covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more sjmthetic amino acids.

8. The composition of claim 1 wherein labetalol is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW243P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein labetalol is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing labetalol from said composition in a pH-dependent manner.

19. A method for protecting labetalol from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of labetalol from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching labetalol to said polypeptide.

21. A method for delivering labetalol to a patient comprising administering to

■ said patient a composition comprising: a polypeptide; and labetalol covalently attached to said polypeptide.

22. The method of claim 21 wherein labetalol is released from said composition by an enzyme-catalyzed release.

17 CW243P

23. The method of claim 21 wherein labetalol is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW243P

Abstract

A composition comprising a polypeptide and labetalol covalently attached to the polypeptide. Also provided is a method for delivery of labetalol to a patient comprising administering to the patient a composition comprising a polypeptide and labetalol covalently attached to the polypeptide. Also provided is a method for protecting labetalol from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of labetalol from a composition comprising covalently attaching it to the polypeptide.

19 CW244P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING LAMIVUDINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to lamivudine, as well as methods for protecting and administering lamivudine. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its phaonaceutical effectiveness.

BACKGROUND OF THE INVENTION

Lamivudine is a known phaonaceutical agent that is used in the treatment of hepatitis, viral infection and HIV infection. Its chemical name is (2R-cis)-4-amino-l-[2- (hydroxymethyl)-l,3-oxathiolan-5-yl]-2(lH)-pyrimidinone. Its stracture is:

The novel phaimaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active phaonaceutical agent, an adjuvant, or an inhibitor. CW244P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW244P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW244P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brash-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (lamivudine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching lamivudine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising lamivudine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and lamivudine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Lamivudine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW244P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-terminus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting lamivudine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering lamivudine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, lamivudine is released from the composition by an enzyme-catalyzed release. In another prefened embodiment, lamivudine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefened embodiment, the composition further comprises a microencapsulating agent and lamivudine is released from the composition by dissolution of the microencapsulating agent. In another prefened embodiment, lamivudine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, lamivudine is released from the composition in a CW244P

sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising α polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching lamivudine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, lamivudine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutaπuc acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutaπuc acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is CW244P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize lamivudine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of lamivudine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Lamivudine is the subject of U.S. Patent Numbers 5,047,407 and 5,905,082, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises lamivudine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino CW244P

acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's CW244P

decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of CW244P

active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) ' 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent

10 CW244P

delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drug is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit fooning a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide canier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide canier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using

11 CW244P

any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tettagastrin and pentagastrin: As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, lamivudine is covalently attached to the polypeptide via the hydroxyl group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW244P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-lamivudine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW244P

AciάVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiiπύde or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW244P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent. ^'

Preparation of Poly [γ- Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW244P

What is claimed is:

1. A phaimaceutical composition comprising: a polypeptide; and lamivudine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein lamivudine is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW244P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein lamivudine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing lamivudine from said composition in a pH-dependent manner.

19. A method for protecting lamivudine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of lamivudine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching lamivudine to said polypeptide.

21. A method for delivering lamivudine to a patient comprising administering to said patient a composition comprising: a polypeptide; and lamivudine covalently attached to said polypeptide.

22. The method of claim 21 wherein lamivudine is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein lamivudine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

« 25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW244P

Abstract

A composition comprising a polypeptide and lamivudine covalently attached to the polypeptide. Also provided is a method for delivery of lamivudine to a patient comprising administering to the patient a composition comprising a polypeptide and lamivudine covalently attached to the polypeptide. Also provided is a method for protecting lamivudine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of lamivudine from a composition comprising covalently attaching it to the polypeptide.

19 CW245P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING LAMIVUDINE AND ZIDOVUDINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to lamivudine and zidovudine, as well as methods for protecting and administering lamivudine and zidovudine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective phaonaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Lamivudine is a known pharmaceutical agent that is used in the treatment of hepatitis, viral infection and HIV infection. Its chemical name is (2R-cis)-4-amino-l-[2- (hydroxymethyl)-l,3-oxathiolan-5-yl]-2(lH)-pyrimidinone. Its stracture is:

Zidovudine has the chemical name 3'-azido-3'-deoxythymidine. Its stracture is

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The two drags together are used as a fixed-dose combination tablet comprising the reverse transcriptase inhibitors lamivudine and zidovudine for the treatment of HIN infection.

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and CW245P

aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders CW245P t and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (lamivudine and zidovudine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching lamivudine and zidovudine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refened to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism. CW245P

Alternatively, the present invention provides a pharmaceutical composition comprising lamivudine and zidovudine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and lamivudine and zidovudine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Lamivudine and zidovudine preferably is covalently attached to a side chain, the

N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the poljφeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner. CW245P

The invention also provides a method for protecting lamivudine and zidovudine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering lamivudine and zidovudine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefened embodiment, lamivudine and zidovudine are released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, lamivudine and zidovudine are released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and lamivudine and zidovudine are released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, lamivudine and zidovudine are released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, lamivudine and zidovudine are released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching lamivudine and zidovudine to a side chain of an amino acid to form an active agent/amino acid complex; (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second CW245P

agent, lamivudine and zidovudine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize lamivudine and zidovudine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of lamivudine and zidovudine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Lamivudine is the subject of U.S. Patent Numbers 5,047,407 and 5,905,082, herein incoφorated by reference, which describes how to make that drag. Zidovudine is covered by EP 196185 B (1989). The combination is covered by the following U.S. CW245P

patents, which are hereby incoφorated by reference:4,724,232, 4,818,538, 4,828,838, 4,833,130, 4,837,208 and 6,113,920.

The composition of the invention comprises lamivudine and zidovudine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with CW245P

protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drugs would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will CW245P

ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the

10 CW245P

jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order

1 1 CW245P

to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide canier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly fooned free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated,

12 CW245P

preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, lamivudine and zidovudine are covalently attached to the polypeptide via the hydroxyl group on each.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known

13 CW245P

intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-lamivudine and zidovudine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

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The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

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The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW245P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and lamivudine and zidovudine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein lamivudine and zidovudine are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein lamivudine and zidovudine are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing lamivudine and zidovudine from said composition in a pH-dependent manner.

19. A method for protecting lamivudine and zidovudine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of lamivudine and zidovudine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching lamivudine and zidovudine to said polypeptide.

21. A method for delivering lamivudine and zidovudine to a patient comprising administering to said patient a composition comprising: a polypeptide; and lamivudine and zidovudine covalently attached to said polypeptide.

22. The method of claim 21 wherein lamivudine and zidovudine are released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein lamivudine and zidovudine are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and lamivudine and zidovudine covalently attached to the polypeptide. Also provided is a method for delivery of lamivudine and zidovudine to a patient comprising administering to the patient a composition comprising a polypeptide and lamivudine and zidovudine covalently attached to the polypeptide. Also provided is a method for protecting lamivudine and zidovudine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of lamivudine and zidovudine from a composition comprising covalently attaching it to the polypeptide.

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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING LAMOTRIGINE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to lamotrigine, as well as methods for protecting and administering lamotrigine. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a caoier compound that enhances the usefulness of the phaonaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Lamotrigine is a known phaimaceutical agent that is used in the treatment of epilepsy, psychosis and depression. Its chemical name is 6-(2,3-dichlorophenyl)- 1,2,4- triazine-3,5-diamine. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW246P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant- on the water solubility of the active agent. Conversely, water-soluble CW246P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drug conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW246P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(lamotrigine) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching lamotrigine to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising lamotrigine microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and lamotrigine covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Lamotrigine preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW246P

the C-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The πucroencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting lamotrigine from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering lamotrigine to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, lamotrigine is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, lamotrigine is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and lamotrigine is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, lamotrigine is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, lamotrigine is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant CW246P

from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching lamotrigine to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, lamotrigine and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW246P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize lamotrigine and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of lamotrigine. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Lamotrigine is the subject of U.S. Patent Numbers 4,602,017 and 5,698,226, herein incoφorated by reference, which describes how to make that drug.

The composition of the invention comprises lamotrigine covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial anangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW246P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. AU of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW246P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW246P

Dextran is the only polysaccharide known that has been explored as a macromolecular carrier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW246P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW246P

maximum drag loading of the carrier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drug delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Altematively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-teoninus, the C-teoninus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, lamotrigine is covalently attached to the polypeptide via the amino group.

The polypeptide caoier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Altematively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

12 CW246P

invention, a pre-first order release of the active agent is imparted by microencapsulating the canier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-lamotrigine conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW246P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tettahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW246P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW246P

What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and lamotrigine covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein lamotrigine is covalently attached to a side chain, the N-teoninus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW246P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein lamotrigine is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing lamotrigine from said composition in a pH-dependent manner.

19. A method for protecting lamotrigine from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of lamotrigine from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching lamotrigine to said polypeptide.

21. A method for delivering lamotrigine to a patient comprising administering to said patient a composition comprising: a polypeptide; and lamotrigine covalently attached to said polypeptide.

22. The method of claim 21 wherein lamotrigine is released from said composition by an enzyme-catalyzed release.

17 CW246P

23. The method of claim 21 wherein lamotrigine is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

CW246P

Abstract

A composition comprising a polypeptide and lamotrigine covalently attached to the polypeptide. Also provided is a method for delivery of lamotrigine to a patient comprising administering to the patient a composition comprising a polypeptide and lamotrigine covalently attached to the polypeptide. Also provided is a method for protecting lamotrigine from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of lamotrigine from a composition comprising covalently attaching it to the polypeptide.

19 CW247P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING LANSOPRAZOLE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to lansoprazole, as well as methods for protecting and administering lansoprazole. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Lansoprazole is a known pharmaceutical agent that is used in the treatment of ulcer and bacterial infection. Its chemical name is 2-[[[3-methyl-4-(2,2,2- trifluoroethoxy)-2-pyridinyl]methyl]sulfinyl]-lH-benzimidazole. Its stracmre is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor. CW247P

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage CW247P

reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drug is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to tteat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in CW247P

the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent (lansoprazole) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching lansoprazole to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaonaceutical composition comprising lansoprazole microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and lansoprazole covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Lansoprazole preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a CW247P

carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teπninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting lansoprazole from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering lansoprazole to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, lansoprazole is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, lansoprazole is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and lansoprazole is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, lansoprazole is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, lansoprazole is released from the CW247P

composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching lansoprazole to a side chain of an amino acid to form an active agent/amino acid complex;

(b) fooning an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, lansoprazole and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is CW247P

described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference.

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize lansoprazole and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of lansoprazole. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Lansoprazole is the subject of U.S. Patent Number 4628098, 4689333, 5026560,

5045321, 5093132 and 5433959, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises lansoprazole covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary structures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a CW247P

particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions. CW247P

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding.

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding. Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the CW247P

caoier, polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent.

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drug. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin ^' 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular

10 CW247P

weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefeoed point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-teoninus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly fooned free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N-

1 1 CW247P

carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence, maximum drug loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This carrier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1 ,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, lansoprazole is covalently attached to the polypeptide via the amino group.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Altematively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW247P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein ttansport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-lansoprazole conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW247P

AciάVN-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide carrier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-teoninus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide caoier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW247P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW247P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and lansoprazole covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said poljφeptide is a homopolymer of a naturally occurring amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein lansoprazole is covalentiy attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW247P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein, said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein lansoprazole is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing lansoprazole from said composition in a pH-dependent manner.

19. A method for protecting lansoprazole from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of lansoprazole from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching lansoprazole to said polypeptide.

21. A method for delivering lansoprazole to a patient comprising administering to said patient a composition comprising: a polypeptide; and lansoprazole covalently attached to said polypeptide.

22. The method of claim 21 wherein lansoprazole is released from said composition by an enzyme-catalyzed release.

17 CW247P

23. The method of claim 21 wherein lansoprazole is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW247P

Abstract

A composition comprising a polypeptide and lansoprazole covalently attached to the polypeptide. Also provided is a method for delivery of lansoprazole to a patient comprising administering to the patient a composition comprising a polypeptide and lansoprazole covalently attached to the polypeptide. Also provided is a method for protecting lansoprazole from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of lansoprazole from a composition comprising covalently attaching it to the polypeptide.

19 CW248P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING LANSOPRAZOLE, AMOXICILLIN AND CLARITHROMICIN AND METHODS

OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to lansoprazole, amoxicillin and clarithromicin, as well as methods for protecting and administering lansoprazole, amoxicillin and clarithromicin. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the phaonaceutical market, and combining it with a caoier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Lansoprazole, amoxicillin and clarithromicin are used together in the treatment of duodenal ulcer. Lansoprazole's chemical name is 2-[[[3-methyl-4-(2,2,2- trifluoroethoxy)-2-pyridinyl]methyl]sulfinyl]-lH-benzimidazole. Its stracture is:

Th

e stracture of amoxicillin is CW248P

The stracture of clarithromicin is

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of CW248P

cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral CW248P

administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(lansoprazole, amoxicillin and clarithromicin) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching lansoprazole, amoxicillin and clarithromicin to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, CW248P

also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the caoier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the caoier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising lansoprazole, amoxicillin and clarithromicin microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and lansoprazole, amoxicillin and clarithromicin covalently attached to the polypeptide. Preferably, the poljφeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Lansoprazole, amoxicillin and clarithromicin preferably is covalently attached to a side chain, the N-terminus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N- terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C- teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N-teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino CW248P

acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting lansoprazole, amoxicillin and clarithromicin from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering lansoprazole, amoxicillin and clarithromicin to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, lansoprazole, amoxicillin and clarithromicin are released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, lansoprazole, amoxicillin and clarithromicin are released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and lansoprazole, amoxicillin and clarithromicin are released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, lansoprazole, amoxicillin and clarithromicin are released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, lansoprazole, amoxicillin and clarithromicin are released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drag conjugate for biphasic release of active ingredients. CW248P

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching lansoprazole, amoxicillin and clarithromicin to a side chain of an amino acid to form an active agent/amino acid complex; -

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA). In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, lansoprazole, amoxicillin and clarithromicin and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaimaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW248P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, ' the invention can stabilize lansoprazole, amoxicillin and clarithromicin and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of lansoprazole, amoxicillin and clarithromicin. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

The composition of the invention comprises lansoprazole, amoxicillin and clarithromicin covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW248P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW248P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the canier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefened. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the carrier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the carrier peptide and the active agent.

10 CW248P

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drug for colon specific drug delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the caoier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

1 1 CW248P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- teoninus of the oligopeptide is the prefeoed point of attachment. If the active agent is ah amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-teoninus or the N-terminus is the prefeoed point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide caoier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide caoier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly fooned free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

12 CW248P

maximum drug loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, lansoprazole, amoxicillin and clarithromicin are covalently attached to the polypeptide via the hydroxyl and or amino groups.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the

13 CW248P

invention, a pre-first order release of the active agent is imparted by microencapsulating the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-lansoprazole, amoxicillin and clarithromicin conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

14 CW248P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stined at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

15 CW248P

Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

16 CW248P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and lansoprazole, amoxicillin and clarithromicin covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occurring amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein lansoprazole, amoxicillin and clarithromicin are covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

17 CW248P

11. The composition of claim 1 further comprising an adjuvant.

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein lansoprazole, amoxicillin and clarithromicin are conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing lansoprazole, amoxicillin and clarithromicin from said composition in a pH-dependent manner.

19. A method for protecting lansoprazole, amoxicillin and clarithromicin from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of lansoprazole, amoxicillin and clarithromicin from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching lansoprazole, amoxicillin and clarithromicin to said polypeptide.

21. A method for delivering lansoprazole, amoxicillin and clarithromicin to a patient comprising administering to said patient a composition comprising: CW248P

a polypeptide; and lansoprazole, amoxicillin and clarithromicin covalently attached to said polypeptide.

22. The method of claim 21 wherein lansoprazole, amoxicillin and clarithromicin are released from said composition by an enzyme-catalyzed release.

23. The method of claim 21 wherein lansoprazole, amoxicillin and clarithromicin are released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

Abstract

A composition comprising a polypeptide and lansoprazole, amoxicillin and clarithromicin covalently attached to the polypeptide. Also provided is a method for delivery of lansoprazole, amoxicillin and clarithromicin to a patient comprising administering to the patient a composition comprising a polypeptide and lansoprazole, amoxicillin and clarithromicin covalently attached to the polypeptide. Also provided is a method for protecting lansoprazole, amoxicillin and clarithromicin from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of lansoprazole, amoxicillin and clarithromicin from a composition comprising covalently attaching it to the polypeptide.

19 CW249P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING LEFLUNOMIDE AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel phaimaceutical compound that comprises a polypeptide that is preferably covalently attached to leflunomide, as well as methods for protecting and administering leflunomide. This novel compound, refeoed to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Leflunomide is a known phaimaceutical agent that is used in the treatment of rheumatoid arthritis. Its chemical name is 5-methyl-N-[4-(trifluoromethyl)phenyl]-4- isoxazolecarboxamide. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel pharmaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW249P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large anay of active agents in tablet formulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW249P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrag formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drug attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drag delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight caoiers are digested slowly or late, as in the case of naproxen-linked dextran, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW249P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(leflunomide) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching leflunomide to the N-terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a carrier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a phaimaceutical composition comprising leflunomide microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and leflunomide covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Leflunomide preferably is covalently attached to a side chain, the N-teoninus or the C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefeoed embodiment, the active agent is an amine and is covalently attached to CW249P

the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- terminus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting leflunomide from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering leflunomide to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, leflunomide is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, leflunomide is released in a time-dependent manner based on the pharmacokinetics of the enzyme- catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and leflunomide is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, leflunomide is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, leflunomide is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and CW249P

release of the adjuvant from the composition is controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching leflunomide to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and

(c) polymerizing the active agent/amino acid complex N-carboxyanhydride (NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, leflunomide and a second active agent can be copolymerized in step (c). In another prefened embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutaπuc acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active phaonaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW249P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize leflunomide and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of leflunomide. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention a o allows targeted delivery of active agents to specifics sites of action.

Leflunomide is the subject of U.S. Patent Number 5679709, herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises leflunomide covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracture of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary stracture.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW249P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperature of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ioeversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW249P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the carrier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine caoy a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary stractures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same carrier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the carrier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW249P

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drag for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drug absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aπύnopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B₂ (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefeoed because confoonational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW249P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit forming a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide carrier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-teoninus of the peptide carrier. Because the glutaπuc acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide caoier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drag moiety from the peptide caoier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drug dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

1 1 CW249P

maximum drag loading of the caoier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drag moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide carrier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

12 CW249P

The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-leflunomide conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

13 CW249P

Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C-terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-teoninus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crude product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

14 CW249P

Preparation of γ-Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drag alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C-Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ- Alkyl Glutamate-NCA

γ-Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

15 CW249P

What is claimed is:

1. A pharmaceutical composition comprising: a polypeptide; and leflunomide covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is' a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein leflunomide is covalently attached to a side chain, the N-terminus or the C-terminus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

16 CW249P

12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein leflunomide is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing leflunomide from said composition in a pH-dependent manner.

19. A method for protecting leflunomide from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for conttolling release of leflunomide from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching leflunomide to said polypeptide.

21. A method for delivering leflunomide to a patient comprising administering to said patient a composition comprising: a polypeptide; and leflunomide covalently attached to said polypeptide.

22. The method of claim 21 wherein leflunomide is released from said composition by an enzyme-catalyzed release.

17 CW249P t

23. The method of claim 21 wherein leflunomide is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

18 CW249P

Abstract

A composition comprising a polypeptide and leflunomide covalently attached to the polypeptide. Also provided is a method for delivery of leflunomide to a patient comprising administering to the patient a composition comprising a polypeptide and leflunomide covalently attached to the polypeptide. Also provided is a method for protecting leflunomide from degradation comprising covalently attaching it to a polypeptide. Also provided is a method for controlling release of leflunomide from a composition comprising covalently attaching it to the polypeptide.

19 CW250P

A NOVEL PHARMACEUTICAL COMPOUND CONTAINING LESOPITRON AND METHODS OF MAKING AND USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical compound that comprises a polypeptide that is preferably covalently attached to lesopitron, as well as methods for protecting and administering lesopitron. This novel compound, refened to as a CARRIERWAVE™ Molecular Analogue (CMA), has the benefit of taking a known effective pharmaceutical agent that is both well studied and occupies a known segment of the pharmaceutical market, and combining it with a carrier compound that enhances the usefulness of the pharmaceutical agent without compromising its pharmaceutical effectiveness.

BACKGROUND OF THE INVENTION

Lesopitron is a known pharmaceutical agent that is used in the treatment of anxiety. Its chemical name is 2-[4-[4-(4-chloro-lH-pyrazol-l-yl)butyl]-l- piperazinyl]pyrimidine dihydrochloride. Its stracture is:

The novel pharmaceutical compound of the present invention is useful in accomplishing one or more of the following goals: enhancement of the chemical stability of the original compound; alteration of the release profile of an orally administered product; enhanced digestion or absoφtion; targeted delivery to particular tissue/cell type; and provision for an oral dosage form when none exists. The novel phaonaceutical compound may contain one or more of the following: another active pharmaceutical agent, an adjuvant, or an inhibitor.

Active agent delivery systems are often critical for the effective delivery of a biologically active agent (active agent) to the appropriate target. The importance of these CW250P

systems becomes magnified when patient compliance and active agent stability are taken under consideration. For instance, one would expect patient compliance to increase markedly if an active agent is administered orally in lieu of an injection or another invasive technique. Increasing the stability of the active agent, such as prolonging shelf life or survival in the stomach, will assure dosage reproducibility and perhaps even reduce the number of dosages required which could improve patient compliance.

Absoφtion of an orally administered active agent is often blocked by the harshly acidic stomach milieu, powerful digestive enzymes in the GI tract, permeability of cellular membranes and transport across lipid bilayers. Incoφorating adjuvants such as resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance permeability of cellular membranes. Microencapsulating active agents using protenoid microspheres, liposomes or polysaccharides have been effective in abating enzyme degradation of the active agent. Enzyme inhibiting adjuvants have also been used to prevent enzyme degradation. Enteric coatings have been used as a protector of pharmaceuticals in the stomach.

Active agent delivery systems also provide the ability to control the release of the active agent. For example, formulating diazepam with a copolymer of glutamic acid and aspartic acid enables a sustained release of the active agent. As another example, copolymers of lactic acid and glutaric acid are used to provide timed release of human growth hormone. A wide range of pharmaceuticals puφortedly provide sustained release through microencapsulation of the active agent in amides of dicarboxylic acids, modified amino acids or thermally condensed amino acids. Slow release rendering additives can also be intermixed with a large aoay of active agents in tablet foonulations.

Each of these technologies imparts enhanced stability and time-release properties to active agent substances. Unfortunately, these technologies suffer from several shortcomings. Incoφoration of the active agent is often dependent on diffusion into the microencapsulating matrix, which may not be quantitative and may complicate dosage reproducibility. In addition, encapsulated drags rely on diffusion out of the matrix, which is highly dependant on the water solubility of the active agent. Conversely, water-soluble CW250P

microspheres swell by an infinite degree and, unfortunately, may release the active agent in bursts with little active agent available for sustained release. Furthermore, in some technologies, control of the degradation process required for active agent release is unreliable. For example, an enterically coated active agent depends on pH to release the active agent and, as such, is difficult to control the rate of release.

In the past, use has been made of amino acid side chains of polypeptides as pendant groups to which active agents can be attached. These technologies typically require the use of spacer groups between the amino acid pendant group and the active agent. The peptide-drag conjugates of this class of drag delivery system rely on enzymes in the bloodstream for the release of the drag and, as such, are not used for oral administration. Examples of timed and targeted release of injectable or subcutaneous pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer, to the gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a peptide spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been covalently attached directly to the beta carboxylate of polyaspartic acid without a spacer group. This prodrug formulation was designed as a colon-specific drag delivery system where the drag is released by bacterial hydrolytic enzymes residing in the large intestines. The released dexamethasone active agent, in turn, was targeted to treat large bowel disorders and was not intended to be absorbed into the bloodstream. Yet another technology combines the advantages of covalent drag attachment with liposome formation where the active ingredient is attached to highly ordered lipid films (known as HARs) via a peptide linker. Thus, there has been no drug delivery system, heretofore reported, that incoφorates the concept of attaching an active ingredient to a polypeptide pendant group with its targeted delivery into the bloodstream via oral administration.

It is also important to control the molecular weight, molecular size and particle size of the active agent delivery system. Variable molecular weights have unpredictable diffusion rates and pharmacokinetics. High molecular weight carriers are digested slowly or late, as in the case of naproxen-linked dexttan, which is digested almost exclusively in the colon by bacterial enzymes. High molecular weight microspheres usually have high moisture content which may present a problem with water labile active ingredients. CW250P

Particle size not only becomes a problem with injectable drags, as in the HAR application, but absoφtion through the brush-border membrane of the intestines is limited to less than 5 microns.

SUMMARY OF THE INVENTION

The present invention provides covalent attachment of the active agent

(lesopitron) to a polymer of peptides or amino acids. The invention is distinguished from the above-mentioned technologies by virtue of covalently attaching lesopitron to the N- terminus, the C-terminus or directly to the amino acid side chain of an oligopeptide or polypeptide, also refeoed to herein as a caoier peptide. In certain applications, the polypeptide will stabilize the active agent, primarily in the stomach, through conformational protection. In these applications, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absoφtion by the body by selectively hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces a second order sustained release mechanism.

Alternatively, the present invention provides a pharmaceutical composition comprising lesopitron microencapsulated by a polypeptide.

The invention provides a composition comprising a polypeptide and lesopitron covalently attached to the polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty namrally occurring amino acids, (iii) a heteropolymer of two or more naturally occuoing amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

Lesopitron preferably is covalently attached to a side chain, the N-terminus or the

C-terminus of the polypeptide. In a prefeoed embodiment, the active agent is a carboxylic acid and is covalently attached to the N-terminus of the polypeptide. In another prefened embodiment, the active agent is an amine and is covalently attached to CW250P

the C-teoninus of the polypeptide. In another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the C-teoninus of the polypeptide. In yet another prefeoed embodiment, the active agent is an alcohol and is covalently attached to the N- teoninus of the polypeptide.

The composition of the invention can also include one or more of a microencapsulating agent, an adjuvant and a pharmaceutically acceptable excipient. The microencapsulating agent can be selected from polyethylene glycol (PEG), an amino acid, a sugar and a salt. When an adjuvant is included in the composition, the adjuvant preferably activates an intestinal transporter.

Preferably, the composition of the invention is in the form of an ingestable tablet, an intravenous preparation or an oral suspension. The active agent can be conformationally protected by folding of the polypeptide about the active agent. In another embodiment, the polypeptide is capable of releasing the active agent from the composition in a pH-dependent manner.

The invention also provides a method for protecting lesopitron from degradation comprising covalently attaching it to a polypeptide.

The invention also provides a method for delivering lesopitron to a patient, the patient being a human or a non-human animal, comprising administering to the patient a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. In a prefeoed embodiment, lesopitron is released from the composition by an enzyme-catalyzed release. In another prefeoed embodiment, lesopitron is released in a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed release. In another prefeoed embodiment, the composition further comprises a microencapsulating agent and lesopitron is released from the composition by dissolution of the microencapsulating agent. In another prefeoed embodiment, lesopitron is released from the composition by a pH-dependent unfolding of the polypeptide. In another prefeoed embodiment, lesopitron is released from the composition in a sustained release. In yet another prefeoed embodiment, the composition further comprises an adjuvant covalently attached to the polypeptide and release of the adjuvant from the composition is CW250P

controlled by the polypeptide. The adjuvant can be microencapsulated into a caoier peptide-drag conjugate for biphasic release of active ingredients.

The invention also provides a method for preparing a composition comprising a polypeptide and an active agent covalently attached to the polypeptide. The method comprises the steps of:

(a) attaching lesopitron to a side chain of an amino acid to form an active agent/amino acid complex;

(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA) from the active agent/amino acid complex; and (c) polymerizing the active agent/amino acid complex N-carboxyanhydride

(NCA).

In a prefeoed embodiment, steps (a) and (b) are repeated prior to step (c) with a second active agent. When steps (a) and (b) are repeated prior to step (c) with a second agent, lesopitron and a second active agent can be copolymerized in step (c). In another prefeoed embodiment, the amino acid is glutamic acid and the active agent is released from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and wherein the active agent is released from the glutamic acid by coincident intramolecular transamination. In another prefeoed embodiment, the glutamic acid is replaced by an amino acid selected from the group consisting of aspartic acid, arginine, asparagine, cysteine, lysine, threonine, and serine, and wherein the active agent is attached to the side chain of the amino acid to form an amide, a thioester, an ester, an ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another prefeoed embodiment, the glutamic acid is replaced by a synthetic amino acid with a pendant group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, but are not restrictive, of the invention. The general applications of this invention to other active pharmaceutical agents is described in U.S. Patent Application Serial Number 09/642,820, filed August 22, 2000, incoφorated herein by reference. CW250P

DETAILED DESCRIPTION OF INVENTION

The present invention provides several benefits for active agent delivery. First, the invention can stabilize lesopitron and prevent its digestion in the stomach. In addition, the pharmacologic effect can be prolonged by delayed release of lesopitron. Furthermore, active agents can be combined to produce synergistic effects. Also, absoφtion of the active agent in the intestinal tract can be enhanced. The invention also allows targeted delivery of active agents to specifics sites of action.

Lesopitron is the subject of EP 382637 A (1990), herein incoφorated by reference, which describes how to make that drag.

The composition of the invention comprises lesopitron covalently attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally occuoing amino acids, (iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of one or more naturally occurring amino acids and one or more synthetic amino acids.

Proteins, oligopeptides and polypeptides are polymers of amino acids that have primary, secondary and tertiary stractures. The secondary stracmre of the protein is the local conformation of the polypeptide chain and consists of helices, pleated sheets and turns. The protein's amino acid sequence and the structural constraints on the conformations of the chain determine the spatial aoangement of the molecule. The folding of the secondary stracture and the spatial aoangement of the side chains constitute the tertiary structure.

Proteins fold because of the dynamics associated between neighboring atoms on the protein and solvent molecules. The thermodynamics of protein folding and unfolding are defined by the free energy of a particular condition of the protein that relies on a particular model. The process of protein folding involves, amongst other things, amino acid residues packing into a hydrophobic core. The amino acid side chains inside the protein core occupy the same volume as they do in amino acid crystals. The folded CW250P

protein interior is therefore more like a crystalline solid than an oil drop and so the best model for determining forces contributing to protein stability is the solid reference state.

The major forces contributing to the thermodynamics of protein folding are Van der Waals interactions, hydrogen bonds, electrostatic interactions, configurational entropy and the hydrophobic effect. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. Comparing the energy of amino acid hydrolysis with protein unfolding in the solid reference state, the hydrophobic effect is the dominant force. Hydrogen bonds are established during the protein fold process and intramolecular bonds are formed at the expense of hydrogen bonds with water. Water molecules are "pushed out" of the packed, hydrophobic protein core. All of these forces combine and contribute to the overall stability of the folded protein where the degree to which ideal packing occurs determines the degree of relative stability of the protein. The result of maximum packing is to produce a center of residues or hydrophobic core that has maximum shielding from solvent.

Since it is likely that lipophilic drags would reside in the hydrophobic core of a peptide, it would require energy to unfold the peptide before the drag can be released. The unfolding process requires overcoming the hydrophobic effect by hydrating the amino acids or achieving the melting temperamre of the protein. The heat of hydration is a destabilization of a protein. Typically, the folded state of a protein is favored by only 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at neutral pH and at room temperature requires chemical reagents. In fact, partial unfolding of a protein is often observed prior to the onset of ineversible chemical or conformation processes. Moreover, protein conformation generally controls the rate and extent of deleterious chemical reactions.

Conformational protection of active agents by proteins depends on the stability of the protein's folded state and the thermodynamics associated with the agent's decomposition. Conditions necessary for the agent's decomposition should be different than for protein unfolding. CW250P

Selection of the amino acids will depend on the physical properties desired. For instance, if increase in bulk or Hpophilicity is desired, then the caoier polypeptide will be enriched in the amino acids in the table provided below. Polar amino acids, on the other hand, can be selected to increase the hydrophilicity of the polypeptide.

Ionizing amino acids can be selected for pH controlled peptide unfolding.

Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the stomach, but will ionize upon entry into the intestine. Conversely, basic amino acids, such as histidine, lysine and arginine, ionize in the stomach and are neutral in an alkaline environment.

Other factors such as π-π interactions between aromatic residues, kinking of the peptide chain by addition of proline, disulfide crosslinking and hydrogen bonding can all be used to select the optimum amino acid sequence for a given application. Ordering of the linear sequence can influence how these interactions can be maximized and is important in directing the secondary and tertiary structures of the polypeptide.

Furthermore, amino acids with reactive side chains (e.g., glutamic acid, lysine, aspartic acid, serine, threonine and cysteine) can be incoφorated for attaching multiple active agents or adjuvants to the same caoier peptide. This is particularly useful if a synergistic effect between two or more active agents is desired.

As stated above, variable molecular weights of the caoier compound can have profound effects on the active agent release kinetics. As a result, low molecular weight active agent delivery systems are prefeoed. An advantage of this invention is that chain length and molecular weight of the polypeptide can be optimized depending on the level of conformational protection desired. This property can be optimized in concert with the kinetics of the first order release mechanism. Thus, another advantage of this invention is that prolonged release time can be imparted by increasing the molecular weight of the caoier polypeptide. Another, significant advantage of the invention is that the kinetics of active agent release is primarily controlled by the enzymatic hydrolysis of the key bond between the caoier peptide and the active agent. CW250P

Dextran is the only polysaccharide known that has been explored as a macromolecular caoier for the covalent binding of drug for colon specific drag delivery. Generally, it was only possible to load up to 1/10 of the total drag-dextran conjugate weight with drag. As stated earlier, polysaccharides are digested mainly in the colon and drag absoφtion is mainly limited to the colon. As compared to dextran, this invention has two major advantages. First, peptides are hydrolyzed by any one of several aminopeptidases found in the intestinal lumen or associated with the brush-border membrane and so active agent release and subsequent absoφtion can occur in the jejunum or the ileum. Second, the molecular weight of the carrier molecule can be controlled and, thus, active agent loading can also be controlled.

As a practical example, the following table lists the molecular weights of lipophilic amino acids (less one water molecule) and selected analgesics and vitamins.

TABLE

Amino acid MW Active agent MW

Glycine 57 Acetaminophen 151

Alanine 71 Vitamin B₆ (Pyroxidine) 169

Valine 99 Vitamin C (Ascorbic acid) 176

Leucine 113 Aspirin 180

Isoleucine 113 Ibuprofen 206

Phenylalanine 147 Retinoic acid 300

Tyrosine 163 Vitamin B (Riboflavin) 376

Vitamin D₂ 397

Vitamin E (Tocopherol) 431

Lipophilic amino acids are prefened because conformational protection through the stomach is important for the selected active agents, which were selected based on ease of covalent attachment to an oligopeptide. Eighteen was subtracted from the amino acid's molecular weight so that their condensation into a polypeptide is considered. For example, a decamer of glycine (MW=588) linked to aspirin would have a total molecular weight of 750 and aspirin would represent 24% of the total weight of the active agent delivery composition or over two times the maximum drag loading for dextran. This is only for an N- or C- terminus application, for those active agents attached to pendant

10 CW250P

groups of decaglutamic acid, for instance, a drag with a molecular weight of 180 could conceivably have a loading of 58%, although this may not be entirely practical.

The alcohol, amine or carboxylic acid group of an active agent may be covalently attached to the N-terminus, the C-terminus or the side chain of the oligopeptide or polypeptide. The location of attachment depends somewhat on the functional group selection. For instance, if the active drag is a carboxylic acid (e.g., aspirin) then the N- terminus of the oligopeptide is the prefened point of attachment. If the active agent is an amine (e.g., ampicillin), then the C-terminus is the prefeoed point of attachment in order to achieve a stable peptide linked active agent. In both, the C- and N-terminus examples, the peptide is, in essence, extended by one monomeric unit fooning a new peptide bond. If the active agent is an alcohol, then either the C-terminus or the N-terminus is the prefened point of attachment in order to achieve a stable composition. As in the example above where the alcohol, norethindrone, was covalently attached to poly(hydroxypropylglutamine), an alcohol can be converted into an alkylchloroformate with phosgene. This invention, then, pertains to the reaction of this key intermediate with the N-terminus of the peptide caoier. The active ingredient can be released from the peptide carrier by intestinal peptidases.

The alcohol can be selectively bound to the gamma carboxylate of glutamic acid and then this conjugate covalently attached to the C-terminus of the peptide carrier. Because the glutamic acid-drag conjugate can be considered a dimer, this product adds two monomeric units to the C-terminus of the peptide carrier where the glutamic acid moiety serves as a spacer between the peptide and the drag as shown in Fig. 4. Intestinal enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug moiety from the peptide carrier. The newly formed free amine of the glutamic acid residue will then undergo an intramolecular transamination reaction, thereby, releasing the active agent with coincident formation of pyroglutamic acid as shown in Fig. 5. Alternatively, the glutamic acid-drag dimer can be converted into the gamma ester of glutamic acid N- carboxyanhydride. This intermediate can then be polymerized, as described above, using any suitable initiator as shown in Fig. 4. The product of this polymerization is polyglutamic acid with active ingredients attached to multiple pendant groups. Hence,

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maximum drag loading of the canier peptide can be achieved. In addition, other amino acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to impart specific properties to the drag delivery system.

The invention also provides a method of imparting the same mechanism of action for other polypeptides containing functional side chains. Examples include, but are not limited to, polylysine, polyasparagine, polyarginine, polyserine, polycysteine, polytyrosine, polythreonine and polyglutamine. The mechanism can translate to these polypeptides through a spacer or linker on the pendant group, which is terminated, preferably, by the glutamic acid-drag dimer. This caoier peptide-drag conjugate is distinguished from the prior art by virtue of the fact that the primary release of the drug moiety relies on peptidases and not on esterases. Alternatively, the active agent can be attached directly to the pendant group where some other indigenous enzymes in the alimentary tract can affect release.

The active agent can be covalently attached to the N-terminus, the C-terminus or the side chain of the polypeptide using known techniques. Examples of linking organic compounds to the N-terminus type of a peptide include, but are not limited to, the attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides and 1,3- dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example, there are known techniques for forming peptide linked biotin and peptide linked acridine.

In the present invention, the active agent is covalently attached to the polypeptide via a linker. This linker may be a small molecule containing 2-6 carbons and one or more functional groups (such as amines, amides, alcohols, or acids) or may be made up of a short chain of either amino acids or carbohydrates.

The polypeptide canier can be prepared using conventional techniques. A prefeoed technique is copolymerization of mixtures of amino acid N-carboxyanhydrides. Alternatively, if a specific sequence is desired, a solid state automated peptide synthesizer can be used.

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The addition of stabilizers to the composition has the potential of stabilizing the polypeptide further. Stabilizers such as sugar, amino acids, polyethylene glycol (PEG) and salts have been shown to prevent protein unfolding. In another embodiment of the invention, a pre-first order release of the active agent is imparted by microencapsulating the caoier polypeptide-active agent conjugate in a polysaccharide, amino acid complex, PEG or salts.

There is evidence that hydrophilic compounds are absorbed through the intestinal epithelia efficiently via specialized transporters. The entire membrane transport system is intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one can expect that excitation of the membrane transport system will involve some sort of specialized adjuvant resulting in localized delivery of active agents. There are seven known intestinal transport systems classified according to the physical properties of the transported substrate. They include the amino acid, oligopeptide, glucose, monocarboxic acid, phosphate, bile acid and the P-glycoprotein transport systems and each has its own associated mechanism of transport. The mechanisms can depend on hydrogen ions, sodium ions, binding sites or other cofactors. The invention also allows targeting the mechanisms for intestinal epithelial transport systems to facilitate absoφtion of active agents.

In another embodiment of the invention, the composition includes one or more adjuvants to enhance the bioavailability of the active agent. Addition of an adjuvant is particularly prefeoed when using an otherwise poorly absorbed active agent. Suitable adjuvants, for example, include: papain, which is a potent enzyme for releasing the catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which activate enzymes in the BBM; and bile acids, which have been attached to peptides to enhance absoφtion of the peptides.

Preferably, the resultant peptide-lesopitron conjugate is formulated into a tablet using suitable excipients and can either be wet granulated or dry compressed.

Compositions of the invention are, in essence, the formation of amides from acids and amines and can be prepared by the following examples.

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Acid/N-terminus conjugation

An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine peptide caoier. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, the product precipitated out in ether and purified using gel permeation chromatography (GPC) or dialysis.

Amine/C -terminus conjugation

The peptide caoier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the amine bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Alcohol/N-Terminus Conjugation

In the following example the combination of the alcohol with triphosgene produces a chloroformate, which when reacted with the N-terminus of the peptide produces a carbamate. Pursuant to this, an alcohol bioactive agent can be treated with triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier is then added slowly and the solution stioed at room temperature for several hours. The product is then precipitated out in ether. The crade product is suitably deprotected and purified using GPC.

Other solvents, activating agents, cocatalysts and bases can be used. Examples of other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or chlorinated solvents such as chloroform. Examples of other activating agents include dicyclohexylcarbodiiπύde or thionyl chloride. An example of another cocatalyst is N- hydroxysuccinimide. Examples of bases include pyoolidinopyridine, dimethylaminopyridine, triethylamine or tributylamine.

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Preparation of γ- Alkyl Glutamate

There have been over 30 different γ-alkyl glutamates prepared any one of which may be suitable for the drug alcohol of choice. For example, a suspension of glutamic acid, the alcohol and concentrated hydrochloric acid can be prepared and heated for several hours. The γ-alkyl glutamate product can be precipitated out in acetone, filtered, dried and recrystallized from hot water.

γ-Alkyl Glutamate/C -Terminus Conjugation

The peptide carrier can be dissolved in DMF under nitrogen and cooled to 0°C. The solution can then be treated with diisopropylcarbodiimide and hydroxybenzotriazole followed by the γ-alkyl glutamate bioactive agent. The reaction can then be stioed for several hours at room temperature, the urea by-product filtered off, and the product precipitated out in ether and purified using GPC or dialysis.

Preparation of γ-Alkyl Glutamate-NCA

γ- Alkyl glutamate can be suspended in dry THF where triphosgene is added and the mixture refluxed under a nitrogen atmosphere until the mixture becomes homogenous. The solution can be poured into heptane to precipitate the NCA product, which is filtered, dried and recrystallized from a suitable solvent.

Preparation of Poly[γ-Alkyl Glutamate]

γ- Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of a primary amine can be added to the solution until it becomes viscous (typically overnight). The product can be isolated from the solution by pouring it into water and filtering. The product can be purified using GPC or dialysis.

Although illustrated and described above with reference to specific embodiments, the invention is nevertheless not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

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Claims

CW250PCLAIMS "What is claimed is:

1. A phaonaceutical composition comprising: a polypeptide; and lesopitron covalently attached to said polypeptide.

2. The composition of claim 1 wherein said polypeptide is an oligopeptide.

3. The composition of claim 1 wherein said polypeptide is a homopolymer of a naturally occuoing amino acid.

4. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more naturally occuoing amino acids.

5. The composition of claim 1 wherein said polypeptide is a homopolymer of a synthetic amino acid.

6. The composition of claim 1 wherein said polypeptide is a heteropolymer of two or more synthetic amino acids.

7. The composition of claim 1 wherein said polypeptide is a heteropolymer of one or more naturally occuoing amino acids and one or more synthetic amino acids.

8. The composition of claim 1 wherein lesopitron is covalently attached to a side chain, the N-teoninus or the C-teoninus of said polypeptide.

9. The composition of claim 1 further comprising a microencapsulating agent.

10. The composition of claim 9 wherein said microencapsulating agent is selected from the group consisting of polyethylene glycol (PEG), an amino acid, a sugar and a salt.

11. The composition of claim 1 further comprising an adjuvant.

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12. The composition of claim 11 wherein said adjuvant activates an intestinal transporter.

13. The composition of claim 1 further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 wherein said composition is in the form of an ingestable tablet.

15. The composition of claim 1 wherein said composition is in the form of an intravenous preparation.

16. The composition of claim 1 wherein said composition is in the form of an oral suspension.

17. The composition of claim 1 wherein lesopitron is conformationally protected by folding of said polypeptide about said active agent.

18. The composition of claim 1 wherein said polypeptide is capable of releasing lesopitron from said composition in a pH-dependent manner.

19. A method for protecting lesopitron from degradation comprising covalently attaching said active agent to a polypeptide.

20. A method for controlling release of lesopitron from a composition wherein said composition comprises a polypeptide, said method comprising covalently attaching lesopitron to said polypeptide.

21. A method for delivering lesopitron to a patient comprising administering to said patient a composition comprising: a polypeptide; and lesopitron covalently attached to said polypeptide.

22. The method of claim 21 wherein lesopitron is released from said composition by an enzyme-catalyzed release.

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23. The method of claim 21 wherein lesopitron is released from said composition by a pH-dependent unfolding of said polypeptide.

24. The method of claim 21 wherein said active agent is released from said composition in a sustained release.

25. The method of claim 21 wherein said composition further comprises an adjuvant covalently attached to said polypeptide and wherein release of said adjuvant from said composition is controlled by said polypeptide.

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